DNA 4Y - The structure of DNA

DNA polymerase catalytic subunit structures

"Cartoon" representation of a DNA polymerase - shows the new nucleotide to be added, sitting at the active site Most polymerases have a 'proofreading' activity; a3'-5' exonuclease which removes the most recently added nucleotide if it does not base-pair correctly with the template -shows how 3' end can flip into the Exo active site when a wrong nucleotide is added.

What is DNA for?

- DNA is an information storage medium - stores 'genetic information' in strings of four 'characters' (A, C, G, T) - The information on DNA must be: - read (transcription+translation) - copied (replication)archived (chromatin etc.) - maintained/repaired 'edited' (recombination) DNA- Deoxyribonucleic acid it is deoxy because - which contains one less oxygen-containing hydroxyl group RNA can be an enzyme unlike DNA linear code of strings of info of those 4 characters

two ways that IHF stabilizes the DNA bends

1. IHF inserts a hydrophobic wedge P=proline between two basepairs ~90o kink 2. charge neutralization: IHF surface has many positively charged aa's

a sharp bend in DNA is energetically costly:

1. reduced stacking of flat basepairs (in B-DNA, stacking maximizes hydrophobic contacts of basepairs) 2. increased charge repulsion of phosphates (in straight B-DNA, phosphates charges are all equally distant) these energy costs would usually keep the DNA straight

how many negative charges per turn of the helix

2 strands / 10.5 per turn / 21 in total

IHF bends the DNA at two ~90o kinks

90 degrees kink: v. wide minor groove mostly roll at one bp step u turn in dna / 2 sharp kinks /

reaction

A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create two identical DNA duplexes from a single original DNA duplex. During this process, DNA polymerase "reads" the existing DNA strands to create two new strands that match the existing ones.[1][2][3][4][5][6] These enzymes catalyze the chemical reaction deoxynucleoside triphosphate + DNAn ⇌ pyrophosphate + DNAn+1. DNA polymerase adds nucleotides to the three prime (3')-end of a DNA strand, one nucleotide at a time. Every time a cell divides, DNA polymerases are required to duplicate the cell's DNA, so that a copy of the original DNA molecule can be passed to each daughter cell. In this way, genetic information is passed down from generation to generation. Before replication can take place, an enzyme called helicase unwinds the DNA molecule from its tightly woven form, in the process breaking the hydrogen bonds between the nucleotide bases. This opens up or "unzips" the double-stranded DNA to give two single strands of DNA that can be used as templates for replication in the above reaction.

the hydrophobic effect

A final fundamental interactioncalled the hydrophobic effect is a manifestation of the proper-ties of water. Some molecules (termed nonpolar molecules)cannot participate in hydrogen bonding or ionic interac-tions. The interactions of nonpolar molecules with watermolecules are not as favorable as are interactions betweenthe water molecules themselves. The water molecules incontact with these nonpolar molecules form "cages" aroundthem, becoming more well ordered than water molecules free in solution. However, when two such nonpolar molecules come together, some of the water molecules are released, allowing them to interact freely with bulk water (Figure 1.12). The release of water from such cages is favorable for reasons to be considered shortly. The result is that nonpolar molecules show an increased tendency to associate with one another in water compared with other, less polar and less self-associating, solvents. This tendency is called the hydrophobic effect and the associated interactions are called hydrophobic interactions.

The building blocks of DNA

Adenine (A) Cytosine (C) Guanine (G) Thymine (T) purines/pyrimidines - bases are flat ( double bonded ) numbering of bases = purines

A- form DNA

Condensed form of DNA. Deeper major groove and shallower minor groove. A-Form DNA was originally identified by X-ray diffraction analysis of DNA fibers at 75% relative humidity (Fuller et al., 1965). The structure of A- DNA is shown in Figure 1.16 and the helix parameters are listed in Table 1.3. The grooves are not as deep as in B-DNA, and the bases are much more tilted (to 20°). Another significant difference between A-form DNA and B-form DNA is that the sugar pucker is C3' endo (compared with C2' endo for B-DNA). Does A-DNA exist in biological systems? Runs of homopurine· ho- mopyrimidine DNA sequence [poly(dG) . poly(dC), for example] seem to set up an A-like helix, as determined by characteristic circular dichroism (CD) spectra (Fairall et al., 1989). Therefore, it is reasonable to assume that within a generally B-like DNA molecule, specific regions may exist in an A-DNA form. This would be a function of sequence composition of DNA (see Section E). RNA frequently exists in a double-helical form in transfer RNAs (tRNA), ribosomal RNAs (rRNA), and parts of messenger RNAs (mRNA). Double- stranded RNA forms an A-like helix. The ribose configuration for double- stranded RNA is C3' endo, which is a distinguishing feature of the A-DNA helix.

Pyrimidines

Cytosine and Thymine One of two categories of nitrogen base ring compounds found in DNA and RNA. A six-membered ring containing two nitrogens

What does DNA do?

DNA contains the instructions needed for an organism to develop, survive and reproduce. To carry out these functions, DNA sequences must be converted into messages that can be used to produce proteins, which are the complex molecules that do most of the work in our bodies.Each DNA sequence that contains instructions to make a protein is known as a gene. The size of a gene may vary greatly, ranging from about 1,000 bases to 1 million bases in humans. Genes only make up about 1 percent of the DNA sequence. DNA sequences outside this 1 percent are involved in regulating when, how and how much of a protein is made.

Conformations of the deoxyribose 5-membered ring

DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although, only B-DNA and Z-DNA have been directly observed in functional organisms.[15] The conformation that DNA adopts depends on the hydration level, DNA sequence, the amount and direction of supercoiling, chemical modifications of the bases, the type and concentration of metal ions, and the presence of polyamines in solution.[41] The first published reports of A-DNA X-ray diffraction patterns—and also B-DNA—used analyses based on Patterson transforms that provided only a limited amount of structural information for oriented fibers of DNA.[42][43] An alternative analysis was then proposed by Wilkins et al., in 1953, for the in vivo B-DNA X-ray diffraction-scattering patterns of highly hydrated DNA fibers in terms of squares of Bessel functions.[44] In the same journal, James Watson and Francis Crickpresented their molecular modeling analysis of the DNA X-ray diffraction patterns to suggest that the structure was a double-helix.[10] Although the B-DNA form is most common under the conditions found in cells,[45] it is not a well-defined conformation but a family of related DNA conformations[46] that occur at the high hydration levels present in cells. Their corresponding X-ray diffraction and scattering patterns are characteristic of molecular paracrystals with a significant degree of disorder.[47][48] Compared to B-DNA, the A-DNA form is a wider right-handed spiral, with a shallow, wide minor groove and a narrower, deeper major groove. The A form occurs under non-physiological conditions in partly dehydrated samples of DNA, while in the cell it may be produced in hybrid pairings of DNA and RNA strands, and in enzyme-DNA complexes.[49][50] Segments of DNA where the bases have been chemically modified by methylation may undergo a larger change in conformation and adopt the Z form. Here, the strands turn about the helical axis in a left-handed spiral, the opposite of the more common B form.[51] These unusual structures can be recognized by specific Z-DNA binding proteins and may be involved in the regulation of transcription.[52] A 2020 study concluded that DNA turned right-handed due to ionization by cosmic rays. Three major forms of DNA are double stranded and connected by interactions between complementary base pairs. These are terms A-form, B-form,and Z-form DNA

DNA resists bending and twisting (or untwisting)

DNA in cells is dynamic and flexible ...in general, any big deformation of the stable B-DNA structure is energetically unfavourable (ie. will not happen spontaneously) the energy 'price' (DG) for bending the DNA must be 'paid' from• interactions with proteins • supercoiling energy, etc • thermal motion/energy bending eg. on nucleosomes twisting eg. in negative supercoiling

DNA structure

DNA is a long polymer made from repeating units called nucleotides, each of which is usually symbolized by a single letter: either A, T, C, or G.[7][8] The structure of DNA is dynamic along its length, being capable of coiling into tight loops and other shapes.[9] In all species it is composed of two helical chains, bound to each other by hydrogen bonds. Both chains are coiled around the same axis, and have the same pitch of 34 angstroms (Å) (3.4 nanometres). The pair of chains has a radius of 10 angstroms (1.0 nanometre).[10] According to another study, when measured in a different solution, the DNA chain measured 22 to 26 angstroms wide (2.2 to 2.6 nanometres), and one nucleotide unit measured 3.3 Å (0.33 nm) long.[11] Although each individual nucleotide is very small, a DNA polymer can be very large and contain hundreds of millions, such as in chromosome 1. Chromosome 1 is the largest human chromosome with approximately 220 million base pairs, and would be 85 mm long if straightened.[12] DNA does not usually exist as a single strand, but instead as a pair of strands that are held tightly together.[10][13] These two long strands coil around each other, in the shape of a double helix. The nucleotide contains both a segment of the backbone of the molecule (which holds the chain together) and a nucleobase (which interacts with the other DNA strand in the helix). A nucleobase linked to a sugar is called a nucleoside, and a base linked to a sugar and to one or more phosphate groups is called a nucleotide. A biopolymer comprising multiple linked nucleotides (as in DNA) is called a polynucleotide.[14] The backbone of the DNA strand is made from alternating phosphate and sugar groups.[15] The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The sugars are joined together by phosphate groups that form phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. These are known as the 3′-end(three prime end), and 5′-end (five prime end) carbons, the prime symbol being used to distinguish these carbon atoms from those of the base to which the deoxyribose forms a glycosidic bond. Therefore, any DNA strand normally has one end at which there is a phosphate group attached to the 5′ carbon of a ribose (the 5′ phosphoryl) and another end at which there is a free hydroxyl group attached to the 3′ carbon of a ribose (the 3′ hydroxyl). The orientation of the 3′ and 5′ carbons along the sugar-phosphate backbone confers directionality (sometimes called polarity) to each DNA strand. In a nucleic acid double helix, the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. The asymmetric ends of DNA strands are said to have a directionality of five prime end (5′ ), and three prime end (3′), with the 5′ end having a terminal phosphate group and the 3′ end a terminal hydroxyl group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose in DNA being replaced by the alternative pentose sugar ribose in RNA The DNA double helix is stabilized primarily by two forces: hydrogen bonds between nucleotides and base-stacking interactions among aromatic nucleobases. The four bases found in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). These four bases are attached to the sugar-phosphate to form the complete nucleotide, as shown for adenosine monophosphate. Adenine pairs with thymine and guanine pairs with cytosine, forming A-T and G-C base pairs

what is DNA made of

DNA is made of chemical building blocks called nucleotides. These building blocks are made of three parts: a phosphate group, a sugar group and one of four types of nitrogen bases. To form a strand of DNA, nucleotides are linked into chains, with the phosphate and sugar groups alternating.The four types of nitrogen bases found in nucleotides are: adenine (A), thymine (T), guanine (G) and cytosine (C). The order, or sequence, of these bases determines what biological instructions are contained in a strand of DNA. For example, the sequence ATCGTT might instruct for blue eyes, while ATCGCT might instruct for brown.The complete DNA instruction book, or genome, for a human contains about 3 billion bases and about 20,000 genes on 23 pairs of chromosomes.

DNA replication

DNA polymerases are responsible for replicating the DNA. They syn- thesize a new strand of DNA using a preexisting strand as a template. They utilize nucleoside triphosphates adding a 5' mononucleotide to the 3' OH end of the nascent DNA chain. Thus, polymerization occurs in the 5' to 3' direc- tion. Because of the 5' to 3' directionality of the polymerase, one strand of a replication fork, the leading strand, can be replicated continuously. The other strand, the lagging strand, must be replicated in short "Okazaki" pieces. There are three polymerases in Escherichia coli called pol I, pol II, and pol III which are encoded by the polA, dinA, and pol C (or dnaE) genes, respec- tively. In E. coli, pol III is primarily responsible for replication of the chromo- some. pol I is involved in joining the short Okazaki pieces. pol I and pol II are involved in DNA repair functions. In eukaryotic cells there are three nuclear polymerases (a, 13, and 8) and one mitochondrial polymerase 'Y. Polymerase a is believed to replicate the leading strand, 8 may replicate the lagging strand, and 13 is involved in DNA repair. These enzymes and the process of DNA replication are described in more detail by Kornberg and Baker (1992). Primase is a required enzyme because DNA polymerase does not initiate synthesis de novo on a single-stranded DNA template. DNA polymerase must have a primer on which to add 5' mononucleotides. The primer, which must be base-paired to the template, can be a short piece of either DNA or RNA with a 3' OH end. RNA polymerase can begin synthetis de novo of RNA complementary to a strand of DNA. A specialized RNA polymerase called primase, encoded by dnaG in E. coli, makes the primer for synthesis by DNA polymerase III. DNA helicases are proteins that move down the DNA separating the two strands or denaturing the double helix. Helicases require energy from ATP for their activity. Helicases work ahead of DNA polymerase, separating the strands for polymerase. There are helicases with two different polarities, those track along a strand in the 3' to 5' direction and those that track from 5' to 3'. DNA ligase is an enzyme that forms a covalent phosphodiester bond between a 3' OH and 5' P04 at the ends of two polynucleotide chains (fre- quently at a nick in one strand of a double helix). Ligase requires energy from ATP to seal the DNA. DNA ligase is used extensively in DNA cloning methodologies

How are DNA sequences used to make proteins?

DNA's instructions are used to make proteins in a two-step process. First, enzymes read the information in a DNA molecule and transcribe it into an intermediary molecule called messenger ribonucleic acid, or mRNA.Next, the information contained in the mRNA molecule is translated into the "language" of amino acids, which are the building blocks of proteins. This language tells the cell's protein-making machinery the precise order in which to link the amino acids to produce a specific protein. This is a major task because there are 20 types of amino acids, which can be placed in many different orders to form a wide variety of proteins

denaturation

Denaturation and renaturation of DNA. The double helical configuration of DNA can be denatured by heat, alkali, or acid. In this process the two strands separate as base stacking and hydrogen bonding interactions are disrupted. Ifone quickly removes the heat or neutralizes the DNA solution (quick cool or neutralize), the DNA will collapse into a compact random coil in which some bases are hydrogen bonded. If a denatured solution of DNA is slowly cooled, the two single strands can reform a paired double helical molecule. The process requires a nucleation event in which a region of complementary bases on opposite strands finds each other, comes into register, and begins to form a hydrogen bonded double helix with stacked base pairs. Once nucleation has occurred, the rest of the DNA molecule rapidly renatures.

Dimensions of 'ideal' B-DNA

Deoxyribose 'sugars' are in C-2'-endo- conformation Bases are in anti-conformation All basepairs are Watson-Crick Helix pitch is the length of one complete helical turn of DNA. In text- book Bform DNA, one helical turn of 10 bp is completed in 34 A. Base pair tilt refers to the angle of the planar bases with respect to the helical axis. The tilt angle is measured by considering the angle made by a line drawn through the two hydrogen bonded bases relative to a line drawn perpendicular to the helix axis. This is illustrated in Figures 1.12, 1.13, and 1.14. A base pair that was perfectly flat, that is, perpendicular to the helix axis, would have a tilt angle of 0°. In Bcform DNA the bases are tilted by only- 6°.In A·form DNA the base pairs are significantly tilted at an angle of 20°

'Sliding clamps' of DNA polymerases

E. coli PolIII is highly processive (i.e., puts on many thousands of nucleotides without falling off the DNA). Processivity requires the 'sliding clamp' β-subunit (as a dimer). stick to each other at two surfaces that makes a hole that dna will pass and DNA polymerase around // tie polymerase on DNA so it docent escape // β2 has a doughnut/polomint-like structure. It seems to be designed not to make any specific contacts with the DNA as it slides along it. In mammalian polymerases, the sliding clamp is PCNA ('proliferating cell nuclear antigen') which is a trimer, but its structure is very similar to the β2 dimer!

Replication of leading and lagging strands (E. coli)

Each PolIII holoenzyme comprises two polymerase functional units; one makes the leading strand continuously, the other makes the lagging strand discontinuously in 1 - 2 kbp Okazaki fragments. replisome contains 2 polymerase functional units - leading and lagging strand holds on the black beta dimer // the clamp // polymerase adds nucleotides //

Structure of β2 dimer + DNA:

Each β subunit has 3 almost identical domains, and each domain is roughly 2-fold symmetric. The hole is lined with 12 α-helices; DNA fits easily into it. To load the sliding clamp (β2) onto DNA, the ring has to be split open. This is done by the γ-complex, and requires ATP hydrolysis. Details are described in Pomerantz et al. 2007. dna in the middle of the dimer ring alpha helices - smooth surfaces how? // rung has to be split the dimers to let dna inside it and that's done by gamma complex and it requires atp hydrolysis

B-DNA and A-DNA

Early structural studies identified two slightly different forms of DNA double helix, A and B.DNA in cells is mostly much like B-DNA (as on the previous slides). A-DNA is a bit 'shorter and fatter', and major and minor grooves are more equal in width than in B-DNA. Some sequences of natural DNA might look like this, or somewhere between B and A. [For most purposes it's simplest to regard B-DNA as the 'standard' DNA structure. On Day 2 we'll discuss how real DNA deviates from ideal B-DNA.]

The double helix is an expression of the rules of chemistry

First, each phosphate group in a DNA strand carries a negative charge. These negatively charged groups interact unfavorably with one another over dis- tances. Thus, unfavorable ionic interactions take place when two strands of DNA come together. These phosphate groups are far apart in the double helix with distances greater than 10 Å, but many such interactions take place (Figure 1.13). Thus, ionic interactions oppose the formation of the double helix. The strength of these repulsive ionic interactions is dimin- ished by the high dielectric constant of water and the presence of ionic species such as Na or Mg2 ions in solution. These positively charged species interact with the phosphate groups and partly neutralize their negative charges. Second, as already noted, hydrogen bonds are important in determining the formation of specific base pairs in the double helix. However, in single- stranded DNA, the hydrogen-bond donors and acceptors are exposed to solution and can form hydrogen bonds with water molecules. When two single strands come together, these hydrogen bonds with water are broken and new hydrogen bonds between the bases are formed. Because the number of hydrogen bonds broken is the same as the number formed, these hydrogen bonds do not contribute substantially to driving the overall process of double-helix formation. However, they contribute greatly to the specificity of binding. Suppose two bases that cannot form Watson-Crick base pairs are brought together. Hydrogen bonds with water must be bro- ken as the bases come into contact. Because the bases are not complemen- tary in structure, not all of these bonds can be simultaneously replaced by hydrogen bonds between the bases. Thus, the formation of a double helix between noncomplementary sequences is disfavored. Third, within a double helix, the base pairs are parallel and stacked nearly on top of one another. The typical separation between the planes of adjacent base pairs is 3.4 Å, and the distances between the most closely approaching atoms are approximately 3.6 Å. This separation distance cor- responds nicely to the van der Waals contact distance (Figure 1.14). Bases tend to stack even in single-stranded DNA molecules. However, the base stacking and associated van der Waals interactions are nearly optimal in a double-helical structure. Fourth, the hydrophobic effect also contributes to the favorability of base stacking. More-complete base stacking moves the nonpolar surfaces of the bases out of water into contact with each other. The principles of double-helix formation between two strands of DNA apply to many other biochemical processes. Many weak interactions con- tribute to the overall energetics of the process, some favorably and some unfavorably. Furthermore, surface complementarity is a key feature: when complementary surfaces meet, hydrogen-bond donors align with hydrogen- bond acceptors and nonpolar surfaces come together to maximize van der Waals interactions and minimize nonpolar surface area exposed to the aque- ous environment. The properties of water play a major role in determining the importance of these interactions

IHF, a well-studied architectural DNA bending protein

IHF (Integration Host Factor)an E. coli protein needed for phage lambda ( l ) integration with many other roles in transcription regulation, DNA compaction lambda integration : IHF + integrase ( a tyrosine recombinase ) lamda excision: IHF + xis + integrase DNA bending by proteins appears to be the rule rather than the exception with the topological distortions ranging from tens of degrees to nearly 720° in the nucleosome core (1, 2). Bending is important in DNA packaging and in regulating diverse cellular processes. The Escherichia coli integration host factor (IHF), discovered as a host protein required for lysogeny by bacteriophage λ, plays both of these roles (3, 4). IHF is an excellent model system for analyzing the mechanism by which proteins bend DNA by virtue of its cellular functions, sequence specificity, tight binding, and robust protein-induced DNA bending (Fig. 1 and refs. 5 and 6). The studies of Sugimura and Crothers (7) and Ansari and coworkers (8) in a recent issue of PNAS directly follow the time evolution of DNA bending upon IHF binding to show that bending sequentially follows protein binding. IHF is a member of a family of Eubacterial proteins that dramatically bend DNA. Although some family members are sequence nonspecific in their binding, IHF binds to specific DNA sequences with nanomolar affinity. DNA structure is important in site selection by all of the family members: DNA bends, nicks, and kinks facilitate binding (6, 10). IHF is a 22-kDa heterodimer of two similar subunits that fold around one another to form a single, compact "body" from which two long β ribbon "arms" extend (Fig. 1). The DNA and protein engage in a mutual embrace: the DNA wraps around the body of the protein whose arms in turn wrap around the minor groove of the DNA. The IHF-induced DNA bend appears to be stabilized by two mechanisms. First, IHF lines the inside face of the DNA with positive charge, allowing bending by mutual repulsion of the phosphates on the outside face of the DNA. Second, the β ribbon arms reach around to the outside of the complex, where the prolines at their tips intercalate between base pairs, compensating for the disruption in stacking present in the tight bend. The bound DNA entails three relatively straight segments separated by the two large kinks where the proline residues are intercalated The "U-turn" bend introduced by IHF brings sequences distant along the DNA duplex into spatial proximity (Fig. 1). This geometry consolidates signals in the primary sequence and could facilitate the cooperative binding of regulatory proteins by means of an "indirect" mechanism. Such a mechanism is observed between IHF and the gpNu1 subunit of λ terminase, the viral DNA packaging enzyme. IHF and gpNu1 bind to cos, the packaging initiation site of λ DNA (Fig. 2). IHF-induced bending at the I1 site juxtaposes the two gpNu1 half-sites, facilitating binding. Conversely, binding of a gpNu1 dimer at R3 and R2 introduces a strong bend into the duplex at I1, facilitating IHF binding (Fig. 2and ref. 9). Cooperative binding of IHF and gpNu1 is thus mediated exclusively by each protein providing a "prebent" architecture without direct interaction. This example illustrates a general mechanism for the assembly of multicomponent nucleoprotein complexes

Deoxyribose Sugar Is Found in DNA

In DNA a slightly different sugar, f3-o-2-deoxyribose, is found. This is a derivative of f3-o-ribose in which the hydroxyl (-OH) at the 2' position is re- placed by a hydrogen (-H). Biochemically this is done by the enzyme ribonu- cleotide reductase which converts all ribonucleoside diphosphates (or occa- sionally triphosphates) in a chemical reduction reaction from 2' OH to 2' H. The sugar moiety of DNA is one of the more flexible and dynamic parts of the molecule. Figure 1.4 shows the structures of the common sugar confor- mations that are found in the various forms of DNA. The sugar ring struc- ture is easy to envision if one thinks of an envelope. In the envelope form, the four carbons form a plane at the corners of the body of the envelope. The oxygen is at the position representing the top of the envelope flap. The oxy- gen can be bent out of the plane of the body of the envelope. Twisting the C2' and C3' carbons relative to the other atoms results in various twist forms of the sugar ring. To form the C2' endo form of the ribose sugar, C2' twists up from the plane of the four carbons. To form the C3' endo, C3' twists down out of the plane of the four carbons. Dideoxyribonucleotides are used in DNA sequencing reactions. A 2',3' dideoxyribonucleotide has hydrogen atoms at both the 2' and 3' positions (see Figure 1.5). When incorporated into a DNA chain, the dideoxyribonu- cleotide blocks further polymerization, since there is no 3' OH to which an- other base can be added. This will become apparent from the ensuing discus- sion of the phosphodiester bond and polynucleotides

Conformation at the glycosidic bond

In DNA, refers to the nitrogen-carbon linkage between the 9' nitrogen of purine bases or 1' nitrogen of pyrimidine bases and the 1' carbon of the sugar group.

bacterial dna polymerase

In E. coli, the main DNA polymerase is PolIII, a large complex with 18 protein subunits. Another important E. coli DNA polymerase, PolI, is mainly used for DNA repair. The PolIII 'replisome' replicates the circular E. coli chromosome. It starts at the 'origin of replication' oriC, a specific place in the chromosome DNA. A sum: E. coli chromosome is 4.7 x 106 bp. It's replicated in 40 minutes, i.e. 2400 sec. Replication from oriC is bidirectional, i.e. one PolIII complex sets of in each direction along the DNA. So, the rate of PolIII = 4.7 x 106/2 x 2400 bp replicated per second,= ~1000 bp (or 2000 nt)/second! Eukaryote replication forks are reported to go slower -~1500 nt/minute. This multi-protein + DNA complex is called the 'replisome ter opposite oric / origin is bidirectional and meet at terms region and finish replication

Watson-Crick base pairing

In a DNA double helix, each type of nucleobase on one strand bonds with just one type of nucleobase on the other strand. This is called complementary base pairing. Purines form hydrogen bonds to pyrimidines, with adenine bonding only to thymine in two hydrogen bonds, and cytosine bonding only to guanine in three hydrogen bonds. This arrangement of two nucleotides binding together across the double helix is called a Watson-Crick base pair. DNA with high GC-content is more stable than DNA with low GC-content. A Hoogsteen base pair is a rare variation of base-pairing.[27] As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily. The two strands of DNA in a double helix can thus be pulled apart like a zipper, either by a mechanical force or high temperature.[28] As a result of this base pair complementarity, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. This reversible and specific interaction between complementary base pairs is critical for all the functions of DNA in organisms. A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA and RNA. Dictated by specific hydrogen bonding patterns, "Watson-Crick" base pairs (guanine-cytosine and adenine-thymine)[1] allow the DNA helix to maintain a regular helical structure that is subtly dependent on its nucleotide sequence.[2] The complementary nature of this based-paired structure provides a redundant copy of the genetic informationencoded within each strand of DNA. The regular structure and data redundancy provided by the DNA double helix make DNA well suited to the storage of genetic information, while base-pairing between DNA and incoming nucleotides provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase transcribes DNA into RNA. Many DNA-binding proteins can recognize specific base-pairing patterns that identify particular regulatory regions of genes. Intramolecular base pairs can occur within single-stranded nucleic acids. This is particularly important in RNA molecules (e.g., transfer RNA), where Watson-Crick base pairs (guanine-cytosine and adenine-uracil) permit the formation of short double-stranded helices, and a wide variety of non-Watson-Crick interactions (e.g., G-U or A-A) allow RNAs to fold into a vast range of specific three-dimensional structures. In addition, base-pairing between transfer RNA (tRNA) and messenger RNA(mRNA) forms the basis for the molecular recognition events that result in the nucleotide sequence of mRNA becoming translated into the amino acid sequence of proteins via the genetic code. The size of an individual gene or an organism's entire genome is often measured in base pairs because DNA is usually double-stranded. Hence, the number of total base pairs is equal to the number of nucleotides in one of the strands (with the exception of non-coding single-stranded regions of telomeres). The haploid human genome (23 chromosomes) is estimated to be about 3.2 billion bases long and to contain 20,000-25,000 distinct protein-coding genes.[3][4][5] A kilobase (kb) is a unit of measurement in molecular biology equal to 1000 base pairs of DNA or RNA.[6] The total number of DNAbase pairs on Earth is estimated at 5.0×1037 with a weight of 50 billion tonnes.[7] In comparison, the total mass of the biosphere has been estimated to be as much as 4 TtC (trillion tons of carbon) (There are other kinds of basepairs, with H-bonds between different atoms in the purines/pyrimidines. These are only found in unusual DNA structures; for example, Hoogsteen basepairs, found in quadruplex DNA etc. But we won't be saying much about them in this course.)

Minor groove

In a helix, refers to the smaller of the unequal grooves that are formed as a result of the double-helical structure of DNA. As a result of the patterns of hydrogen bonding between complementary bases of DNA, the sugar groups stick out at 120 degree angles from each other instead of 180. The minor groove is generated by the smaller angular distance between sugars.

at the replication fork

It's probably most realistic to think of the proteins as being fixed in one place in the cell, so that the DNA is being passed through a stationary replication complex

Deoxyribonucleotides

Nucleotides containing 2-deoxy-D-ribose as the pentose component. A 'chemist's' drawing of a nucleotide, showing the conventional numbering of the deoxyribose, and where the 5' and 3' phosphates are attached in DNA - oxygen - deoxyribose contains 5 carbon atoms to distinguish the atoms from the base, there's a priming numbering system 5'C - not part of the ring that's attached to a phosphate 3'c - important positions / chain carries on from this point

Purines

Purine One of two categories of nitrogen base ring compounds found in DNA and RNA. A purine is a nine-membered double ring composed of one five-membered joined to a six membered ring containing four nitrogens

A brief summary on structural variation

Real DNA is almost never exactly like the 'ideal' B structure. Its character isaffected by its basepair sequence, and by external factors like ions and supercoiling. Examples: The width of the minor groove varies (e.g. GC-rich wide, AT-rich narrow) Helix twist per basepair varies quite a lot Basepairs can be distorted, and don't always sit flat on each other The DNA axis can bend etc. These variations have important implications for genome structure and stability, and for protein-DNA interactions. More about these issues on Day 2! AND... Cellular DNA is usually covered with DNA-binding proteins! Binding of proteins can change DNA structure dramatically. More on Days 3 and 4

Anti-parallel

Refers to the orientations of the two single strands that compose a double-stranded DNA helix. Strands are oriented such that one strand's 5' end is directly across from the other strand's 3' end.

What is the DNA double helix?

Scientist use the term "double helix" to describe DNA's winding, two-stranded chemical structure. This shape - which looks much like a twisted ladder - gives DNA the power to pass along biological instructions with great precision.To understand DNA's double helix from a chemical standpoint, picture the sides of the ladder as strands of alternating sugar and phosphate groups - strands that run in opposite directions. Each "rung" of the ladder is made up of two nitrogen bases, paired together by hydrogen bonds. Because of the highly specific nature of this type of chemical pairing, base A always pairs with base T, and likewise C with G. So, if you know the sequence of the bases on one strand of a DNA double helix, it is a simple matter to figure out the sequence of bases on the other strand.DNA's unique structure enables the molecule to copy itself during cell division. When a cell prepares to divide, the DNA helix splits down the middle and becomes two single strands. These single strands serve as templates for building two new, double-stranded DNA molecules - each a replica of the original DNA molecule. In this process, an A base is added wherever there is a T, a C where there is a G, and so on until all of the bases once again have partners. In addition, when proteins are being made, the double helix unwinds to allow a single strand of DNA to serve as a template. This template strand is then transcribed into mRNA, which is a molecule that conveys vital instructions to the cell's protein-making machinery.

IHF protein induces a DNA bend of ~ 180o

Slide 1 from a crystal structure of IHF bound to a specific DNA sequence (more about sequnce specificity on day 3)

Eukaryotes

The 'core' eukaryote replisome contains more than 30 different proteins, but many more proteins join in as replication proceeds. There are at least 3 polymerase enzymes at the replication fork (each enzyme is a tetramer of four different subunits). Polα (includes primase, and extends RNA primers ~10-12 nt); Polε (makes leading strand)Polδ (makes lagging strand) The specific functions of each Pol in replication are still quite controversial In total, humans have about 20 different DNA polymerases! Others Many phages, viruses and transposons encode their own DNA polymerases. And we mustn't forget reverse transcriptase - a DNA polymerase that uses RNA as a template. more extra proteins that join while various things are happening / time wise //

The building blocks of DNA

The DNA double helix is made of two antiparallel, base-paired strands. Each strand has the structure shown here "The uniqueness of a given DNA structure lies solely in the sequence of its bases." each of these strands has the chemical structure// each unit looks exactly the same except for the base ( can be either C,A,T,G) // no base ( backbone which is sugar and phosphate ) - millions of nucleotides sequence is bases is what's unique

Major and minor groove

The DNA forms 2 grooves. most proteins bind to the major groove. The major groove occurs where the backbones are far apart, the minor groove occurs where they are close together. The major groove is wider than the minor groove in DNA , and many sequence specific proteins interact in the major groove. The N7 and C6 groups of purines and the C4 and C5 groups of pyrimidines face into the major groove, thus they can make specific contacts with amino acids in DNA-binding proteins. Thus specific amino acids serve as H‑bond donors and acceptors to form H-bonds with specific nucleotides in the DNA. H‑bond donors and acceptors are also in the minor groove, and indeed some proteins bind specifically in the minor groove. Base pairs stack, with some rotation between them

2. Denaturation and Renaturation

The double-helical structure of DNA is remarkably stable. This stability is derived from two chemical forces, hydrogen bonding and base stacking in- teractions, as discussed in Section C,1. In addition, the helix is solvated or covered with water molecules which form a "shell of hydration" around the DNA. To melt the two strands or denature the DNA, all these stabilizing forces must be overcome. The two strands of DNA come apart readily on incubation at pH > 12 or pH < 2 due to ionization of the bases (Figure 1.19). As discussed earlier, ionization results in a change in the hydrogen bond donor/acceptor properties of the bases, which will disrupt the normal A . T and C . G Watson-Crick hy- drogen bonds. In addition, the shell of hydration surrounding the DNA is dis- rupted at very high or low pH, destabilizing base stacking. Acid treatment of DNA leads to depyrimidation and depurination, the loss of bases by cleavage of the glycosidic bond. The phosphodiester bond is more susceptible to hy- drolysis at these abasic sites. Thus, strong acid will degrade DNA. Since sin- gle strands of DNA are relatively stable in alkali, denaturation is usually ac- complished by alkali treatment. Increasing the temperature of DNA destabilizes the double helix, result- ing in the separation of the two strands. Heat both disrupts the hydrogen bonds and destroys the shell of hydration of DNA leading to a loss of forces holding the two strands together. Experimentally, there is a linear relation- ship between the G + C content and the melting temperature of DNA (Marmur and Doty, 1962). The temperature at which 50% of a DNA sample is melted is called the melting temperature or Tm- Moreover, there is a linear relationship between the Tm of DNA and the calculated stacking energies (listed in Table 1.2). This suggests that the thermal stability of DNA is a func- tion of base stacking, not only hydrogen bonding (see Saenger, 1984, for more discussion). DNA denaturation can be measured or monitored in many different ways. One method involves measurement of a characteristic increase in the absorbance at 260 nm called hyperchromicity, which results from the un- stacking of the bases. Another method employs enzymes specific for single- stranded DNA (such as Sl nuclease). Selective binding reactivity of single- or double-stranded DNAs with various surfaces can also be used. For example, hydroxylapatite, a C a P 0 4 precipitate, will bind double-stranded DNA but not single-stranded DNA at certain phosphate concentrations. Rapid removal of denaturing conditions, such as quickly cooling a heat- denatured DNA sample, results in the collapse of single strands into an un-ordered random coil (Figure 1.19). In this configuration the two strands can- not reform a double helix. However, if DNA is slowly cooled, interstrand nucleation can occur in which small complementary regions on the two op- posite DNA strands come together and form a short double-helical region. Once nucleation has occurred, the rest of the double helix renatures very rapidly. The rate limiting step in renaturation is the nucleation event. Negative DNA supercoiling, which is discussed in Chapter 3, can also drive the melting of a region of DNA. Supercoiled DNA contains a great deal of free energy that can be used to transiently or stably melt local regions of supercoiled DNA. Not surprisingly, these are usually regions of DNA rich in A + T.

Conservation of replication proteins

The functions of the proteins in the replisome are conserved through all living organisms (though the proteins may be unrelated). For example: see table imp

B-form DNA

The information from the base composition of DNA, the knowledge of dinucleotide structure, and the insight that the X‑ray crystallography suggested a helical periodicity were combined by Watson and Crick in 1953 in their proposed model for a double helical structure for DNA. They proposed two strands of DNA -- each in a right‑hand helix -- wound around the same axis. The two strands are held together by H‑bonding between the bases (in anti conformation).Bases fit in the double helical model if pyrimidine on one strand is always paired with purine on the other. From Chargaff's rules, the two strands will pair A with T and G with C. This pairs a keto base with an amino base, a purine with a pyrimidine. Two H‑bonds can form between A and T, and three can form between G and C. This third H-bond in the G:C base pair is between the additional exocyclic amino group on G and the C2 keto group on C. The pyrimidine C2 keto group is not involved in hydrogen bonding in the A:T base pair. These are the complementary base pairs. The base‑pairing scheme immediately suggests a way to replicate and copy the the genetic information.The two strands are not in a simple side‑by‑side arrangement, which would be called a paranemic joint (Figure 2.5.32.5.3). (This will be encountered during recombination in Chapter 8.) Rather the two strands are coiled around the same helical axis and are intertwined with themselves (which is referred to as a plectonemic coil). One consequence of this intertwining is that the two strands cannot be separated without the DNA rotating, one turn of the DNA for every "untwisting" of the two strands

Differences between A-form and B-form nucleic acid

The major difference between A-form and B-form nucleic acid is in the conformation of the deoxyribose sugar ring. It is in the C2' endoconformation for B-form, whereas it is in the C3' endoconformation in A-form. , if you consider the plane defined by the C4'-O-C1' atoms of the deoxyribose, in the C2' endoconformation, the C2' atom is above the plane, whereas the C3' atom is above the plane in the C3' endoconformation. The latter conformation brings the 5' and 3' hydroxyls (both esterified to the phosphates linking to the next nucleotides) closer together than is seen in the C2' endoconfromation . Thus the distance between adjacent nucleotides is reduced by about 1 Angstrom in A-form relative to B-form nucleic acid. A second major difference between A-form and B-form nucleic acid is the placement of base-pairs within the duplex. In B-form, the base-pairs are almost centered over the helical axis , but in A-form, they are displaced away from the central axis and closer to the major groove. The result is a ribbon-like helix with a more open cylindrical core in A-form.

discovery of DNA began in 1860's

The molecule now known as DNA was first identified in the 1860s by a Swiss chemist called Johann Friedrich Miescher. Johann set out to research the key components of white blood cells?, part of our body's immune system. The main source of these cells? was pus-coated bandages collected from a nearby medical clinic. Johann carried out experiments using salt solutions to understand more about what makes up white blood cells. He noticed that, when he added acid to a solution of the cells, a substance separated from the solution. This substance then dissolved again when an alkali was added. When investigating this substance he realised that it had unexpected properties different to those of the other proteins? he was familiar with. Johann called this mysterious substance 'nuclein', because he believed it had come from the cell nucleus?. Unbeknown to him, Johann had discovered the molecular basis of all life - DNA. He then set about finding ways to extract it in its pure form. Johann was convinced of the importance of nuclein and came very close to uncovering its elusive role, despite the simple tools and methods available to him. However, he lacked the skills to communicate and promote what he had found to the wider scientific community. Ever the perfectionist, he hesitated for long periods of time between experiments before he published his results in 1874. Before then he primarily discussed his findings in private letters to his friends. As a result, it was many decades before Johann Friedrich Miescher's discovery was fully appreciated by the scientific community. For many years, scientists continued to believe that proteins were the molecules that held all of our genetic material. They believed that nuclein simply wasn't complex enough to contain all of the information needed to make up a genome. Surely, one type of molecule could not account for all the variation seen within species

syn and anti conformation

The plane of the base is almost perpendicular to that of the sugars and approximately bisects the O4'-C1'-C2' angle. This allows the bases to occupy either of two principal orientations. The anti conformer has the smaller H-6 (pirimidine) or H-8 (purine) atom above the sugar ring, while the syn conformer has the larger O-2 (pirimidine) or N-3 (purine) in that position. Pirimidines occupy a narrow range of anti conformations, while purines are found in wider range of anti conformations. There is only one known case where a purine adopts a syn conformation. The unusual form is to find in an even more unusual structure namely left handed Z-DNA. An example of possible interaction between guanosines in syn and anti conformation is shown on a drawing below Two other bonds which are important for determining the DNA structure are: the bond between C4' and C5' which defines the position of the 5'phosphate relative to the sugar ring (the favoured conformations of this bond are synclinical and antiperiplanar) C-O and P-O bonds (the former usually appears in antiperiplanar conformation and the latter in gauche conformation. Syn addition occurs when H2 reacts with a double bond. In this type of a reaction, both hydrogen atoms are added to the same side. The product that puts the hydrogen atoms on opposite sides doesn't form, like you can see here:The reason has to do with the mechanism for adding hydrogen to an alkene. We're actually first plating hydrogens onto a thin metal sheet (the Pd-C). This plating keeps the hydrogen atoms from freely rotating around the molecule, so they must be added to the same side of the molecule

Purines and Pyrimidines as Informational Molecules

The purines and pyrimidines are well suited to their roles as the infor- mational molecules of the cell. The differential placement of hydrogen bond donor and acceptor groups gives the bases the unique structural identity that allows them to serve as the genetic information. The hydrogen atoms of amino groups provide hydrogen bond donors, whereas the carbonyl oxygens and ring nitrogens provide hydrogen bond acceptors. The aromatic nature of the rings means that they are rigid planar molecules. This flatness is impor- tant in the organization of bases within the helix, since it allows the bases to stack uniformly within the helix. As described subsequently, this stacking helps protect the chemical identity of the bases

base pairs

The two Watson-Crick basepairs have very similar sizes and shapes, allowing them to fit in any sequence into a quite uniform double helix structure "major groove" and "minor groove" indicate where the edges of the basepairs end up in the double helix. A-T = space floor representation hydrogen atoms don't show up B- Nitrogen R- oxygen

Denaturation ("melting") of DNA

The two strands of DNA are held together by many weak interactions (mainly H-bonds). The strands can come apart (melt) quite easily. Proteins can do this (e.g. helicases, RNA polymerase etc.), but it can also happen at high temperature, in unusual chemical conditions or by using chemical denaturants such as formamide. "Melting temperature" of DNA depends on the AT/GC content, and salt concentration, pH, etc. The reverse of melting is called annealing

van deer Waals

ThebasisofavanderWaalsinteractionis that the distribution of electronic charge around an atom fluctuates with time. At any instant, the charge distribution is not perfectly symmetric. This transient asymmetry in the electronic charge about an atom acts through ionic interactions to induce a complementary asymmetry in the electron distribution within its neighboring atoms. The atom and its neighbors then attract one another. This attraction increases as two atoms come closer to each other, until they are separated by the van der Waals contact distance (Figure 1.10). At distances shorter than the van der Waals contact distance, very strong repulsive forces become dominant because the outer electron clouds of the two atoms overlap. Energies associated with van der Waals interactions are quite small; typical interactions contribute from 2 to 4 kJ mol1 (from 0.5 to 1 kcal mol1) per atom pair. When the surfaces of two large molecules come together, however, a large number of atoms are in van der Waals contact, and the net effect, summed over many atom pairs, can be substantial.

A 'zoo' of unusual DNA structures

These 'unusual' forms of DNA may be important in some biological events. We'll mention some of them later in the course. At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes.[58] These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected.[59] In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence. These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases, known as a guanine tetrad, form a flat plate. These flat four-base units then stack on top of each other to form a stable G-quadruplex structure.[62] These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit.[63] Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure. In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins.[64] At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop or D-loop. Z-DNA is a left-handed helix that is very different from right-handed DNA forms. Z-DNA can form in alternating purine-pyrimidine tracts under certain conditions, including high salt, the presence of certain divalent cations, or DNA supercoiling. Compared with B-DNA, there are major struc- tural differences in the sugar pucker, rotations about the glycosidic bond, and orientation of base pairs within the helix (Table 1.3)

hydrogen bonds

These interactions are largely ionic interactions, with partial charges on nearby atoms attracting one another. Hydrogen bonds are responsible for specific base-pair formation in the DNA double helix. The hydrogen atom in a hydrogen bond is partially shared by two electronega- tive atoms such as nitrogen or oxygen. The hydrogen-bond donor is the group that includes both the atom to which the hydrogen atom is more tightly linked and the hydrogen atom itself, whereas the hydrogen-bond acceptor is the atom less tightly linked to the hydrogen atom (Figure 1.9). The electro- negative atom to which the hydrogen atom is covalently bonded pulls elec- tron density away from the hydrogen atom, which thus develops a partial positive charge (d). Thus, the hydrogen atom with a partial positive charge can interact with an atom having a partial negative charge (d) through an ionic interaction. Hydrogen bonds are much weaker than covalent bonds. They have ener- gies ranging from 4 to 20 kJ mol1 (from 1 to 5 kcal mol1). Hydrogen bonds are also somewhat longer than covalent bonds; their bond lengths (measured from the hydrogen atom) range from 1.5 Å to 2.6 Å; hence, a distance ranging from 2.4 Å to 3.5 Å separates the two nonhydrogen atoms in a hydrogen bond. The strongest hydrogen bonds have a tendency to be approximately straight, such that the hydrogen-bond donor, the hydrogen atom, and the hydrogen-bond acceptor lie along a straight line. This tendency toward lin- earity can be important for orienting interacting molecules with respect to one another. Hydrogen-bonding interactions are responsible for many of the properties of water that make it such a special solvent, as will be described shortly.

A‑form nucleic acids and Z‑DNA

Three different forms of duplex nucleic acid have been described. The most common form, present in most DNA at neutral pH and physiological salt concentrations, is B-form. That is the classic, right-handed double helical structure we have been discussing. A thicker right-handed duplex with a shorter distance between the base pairs has been described for RNA-DNA duplexes and RNA-RNA duplexes. This is called A-form nucleic acid. A third form of duplex DNA has a strikingly different, left-handed helical structure. This Z DNA is formed by stretches of alternating purines and pyrimidines, e.g. GCGCGC, especially in negatively supercoiled DNA. A small amount of the DNA in a cell exists in the Z form. It has been tantalizing to propose that this different structure is involved in some way in regulation of some cellular function, such as transcription or regulation, but conclusive evidence for or against this proposal is not available yet

Structures of purine and pyrimidine bases.

To understand the structure of B-form DNA and numerous structural variations in the DNA helix, it is important to have an appreciation of the in- dividual components of DNA. DNA is composed of aromatic bases (a purine or pyrimidine ring), ribose sugars, and phosphate groups. The many varia- tions in the structures of the bases and the sugars, and in the structural rela- tionship of the base to the sugar, give rise to differences in the helical struc- ture of DNA Purines: Adenine and Guanine Two different heterocyclic aromatic bases with a purine ring (composed of carbon and nitrogen) are found in DNA. The numbering system for the purine ring is shown in Figure 1.2. The two common purine bases found in DNA, adenine and guanine, are also shown in Figure 1.2. These are synthe- sized in cells de novo in multistep biochemical reactions with the base being built on a phosphorylated ribose sugar molecule. The last common interme- diate in their synthesis is inosine. Adenine has an amino group (-NH2 ) on the C6 position of the purine ring (carbon at position 6 of the purine ring). Guanine has an amino group at the C2 position and a carbonyl group at the C6 position. Pyrimidines: Thymine, Cytosine, and Uracil The two pyrimidine bases commonly found in DNA are thymine and cytosine. These are also synthesized in cells de novo in multistep reactions. The structures of the basic six-member ring, thymine, and cytosine are shown in Figure 1.2. Thymine contains a methyl group at the C5 position with car- bonyl groups at the C4 and C2 positions. Cytosine contains a hydrogen atom at the C5 position and an amino group at C4. Uracil is similar to thymine but lacks the methyl group at the C5 posi- tion (Figure 1.2). Uracil is not usually found in DNA. It is a component of ri- bonucleic acid (RNA) in which it is utilized in place of thymine as one of the pyrimidines. RNA also differs from DNA in the structure of the sugar moi- ety, as described later.

DNA REP

Two crucial features of DNA polymerases:(1) DNA polymerase requires a primer. It will only add nucleotides onto the end of an existing strand; it can't start a new one.(2) DNA polymerase requires a template strand. The nucleotide to be added must be complementary (i.e. make a Watson-Crick basepair) to the next base in the template. reactions that DNA polymerase catalyses

Properties of water

Water is the solvent in which most biochemical reac- tions take place, and its properties are essential to the formation of macro- molecular structures and the progress of chemical reactions. Two properties of water are especially relevant: 1. Water is a polar molecule. The water molecule is bent, not linear, and so the distribution of charge is asymmetric. The oxygen nucleus draws elec- trons away from the two hydrogen nuclei, which leaves the region around each hydrogen atom with a net positive charge. The water molecule is thus an electrically polar structure. 2. Water is highly cohesive. Water molecules interact strongly with one another through hydrogen bonds. These interactions are apparent in the structure of ice (Figure 1.11). Networks of hydrogen bonds hold the structure together; similar interactions link molecules in liquid water and account for many of the properties of water. In the liquid state, approximately one in four of the hydrogen bonds present in ice are broken. The polar nature of water is responsible for its high dielectric constant of 80. Molecules in aqueous solution interact with water mole- cules through the formation of hydrogen bonds and through ionic interactions. These interactions make water a versatile solvent, able to readily dissolve many species, especially polar and charged compounds that can participate in these interactions.

DNA in 1953

Watson and Crick discover that DNA structure is a double helix (Watson won nobel prize but had to resign later for crude comments about african inferiority) The Swiss biochemist Frederich Miescher first observed DNA in the late 1800s. But nearly a century passed from that discovery until researchers unraveled the structure of the DNA molecule and realized its central importance to biology.For many years, scientists debated which molecule carried life's biological instructions. Most thought that DNA was too simple a molecule to play such a critical role. Instead, they argued that proteins were more likely to carry out this vital function because of their greater complexity and wider variety of forms.The importance of DNA became clear in 1953 thanks to the work of James Watson*, Francis Crick, Maurice Wilkins and Rosalind Franklin. By studying X-ray diffraction patterns and building models, the scientists figured out the double helix structure of DNA - a structure that enables it to carry biological information from one generation to the next.

B- FORM DNA

Watson-Crick model DNA. Deep, wide major groove. The structure of B-form DNA, the most common form, was originally deduced from X-ray diffraction analysis of the sodium salt of DNA fibers at 92% relative humidity (Langridge et al., 1960a,b). B-Form DNA is pictured in Figure 1.12, where various helix parameters and features are indicated. A molecular model is shown in Figure 1.14. There are about 10.5 bp per right- handed helical turn in B-DNA (helix parameters are listed in Table 1.3). The form of the ribose sugar is C2' endo. The term B-form DNA will be used to refer to the right-handed helical form commonly found for DNA in solution. A dominant feature of B-form DNA is the presence of two distinct grooves, a major and a minor groove, shown in Figures 1.12 and 1.15. These two grooves obviously provide very distinct surfaces with which proteins can interact. As discussed in Chapter 8, different DNA binding proteins have do- mains that interact with either the major or the minor groove. Certain chemi- cals and drugs can interact specifically with either the major or the minor groove. Different functional groups on the purine and pyrimidine bases are accessible from the major or the minor groove (Figure 1.15). The Watson-Crick hydrogen bonding surfaces are not available to solvent or pro- teins, since the functional groups involved in hydrogen bonding are interact- ing with each other (in complementary base pairs) at the center of the double helix. The Hoogsteen hydrogen bonding surface of purines is accessible through the major groove. This is evident by looking down the axis of the double helix as shown in the representation in Figure 1.14. In this projection, the stacked base pairs form a central core surrounded by the phosphate back- bone. The center of the helix is a relatively chemically inert place to store ge- netic information.

revision

What do we mean by the following terms: template; primer; leading strand; lagging strand; semiconservative replication; replisome; polymerase; helicase; proof-reading? Why does DNA replication need two polymerases at the replication fork, that do different things? What are Okazaki fragments, how do they get made? What is the sliding clamp for, and why does the system need a clamp loader? Why does replication need a helicase and topoisomerases, ahead of the replication fork? What is different between bacterial (e.g. E. coli) and eukaryote DNA replication?

Building the double helix

What holds the DNA in a double helix structure? basepairing of complementary strands basepair stacking phosphate charge repulsion preferred conformations of the deoxyribose and phosphate bonds

Z-form DNA

Z-DNA is a radically different duplex structure, with the two strands coiling in left-handed helices and a pronounced zig-zag (hence the name) pattern in the phosphodiester backbone. As previously mentioned, Z-DNA can form when the DNA is in an alternating purine-pyrimidine sequence such as GCGCGC, and indeed the G and C nucleotides are in different conformations, leading to the zig-zag pattern. The big difference is at the G nucleotide. It has the sugar in the C3' endoconformation (like A-form nucleic acid, and in contrast to B-form DNA) and the guanine base is in the synconformation. This places the guanine back over the sugar ring, in contrast to the usual anticonformation seen in A- and B-form nucleic acid. Note that having the base in the anticonformation places it in the position where it can readily form H-bonds with the complementary base on the opposite strand. The duplex in Z-DNA has to accomodate the distortion of this G nucleotide in the synconformation. The cytosine in the adjacent nucleotide of Z-DNA is in the "normal" C2' endo, anticonformation.Even classic B-DNA is not completely uniform in its structure. X-ray diffraction analysis of crystals of duplex oligonucleotides shows that a given sequence will adopt a distinctive structure. These variations in B-DNA may differ in the propeller twist (between bases within a pair) to optimize base stacking, or in the 3 ways that 2 successive base pairs can move relative to each other: twist, roll, or slide.

Major groove

a groove that spirals around the DNA double helix; provides a location where a protein can bind to a particular sequence of bases and affect the expression of a gene In a helix, refers to the larger of the unequal grooves that are formed as a result of the double-helical structure of DNA. As a result of the patterns of hydrogen bonding between complementary bases of DNA, the sugar groups stick out at 120 degree angles from each other instead of 180. The major groove is generated by the larger angular distance between sugars.

conformation at glycosidic bond

anti-pyrimidine (T) anti-purine (A) syn-pyrimidine (T) syn-purine (A) All the nucleotides in A and B-DNA are anti-

Basepair stacking

because they're flat they are hydrophobic and to escape from water they like to stack together ( minimise exposure of surface of bp to water ) The flat surfaces of the basepairs (i.e. their 'top'and 'bottom') are hydrophobic ('water-hating'). To escape from the water they like to stack on top of each other. they'll come as close as together until van Der waals repels them Base stacking is a common arrangement of nucleobases found in the three dimensional structure of nucleic acids. Bases (or base pairs) are planar, and these planes stack at contact distance (about 3.4 Angstrom), excluding water and maximizing Van der Waals interactions. In terms of structural stability of nucleic acids in aqueous solution, the stacking interactions of bases play a larger role than the hydrogen bonds formed by the bases DNA double helix: In double-stranded DNA, bases from two strands pair up to form base pairs, which are stacked along the helix axis of the double strand. Zooming in to a detailed view of a G:C base pair, the extent of the stacking contacts are determined by the sequence. G:C base pairs contribute more to the thermal stability of DNA than A:T base pairs because they stack better.

bacterial genomes

contain all the information for the structure and functioning of a cell Bacterial genomes are generally smaller and less variant in size among species when compared with genomes of eukaryotes. Bacterial genomes can range in size anywhere from about 130 kbp[1][2] to over 14 Mbp.[3] A study that included, but was not limited to, 478 bacterial genomes, concluded that as genome size increases, the number of genes increases at a disproportionately slower rate in eukaryotes than in non-eukaryotes. Thus, the proportion of non-coding DNA goes up with genome size more quickly in non-bacteria than in bacteria. This is consistent with the fact that most eukaryotic nuclear DNA is non-gene coding, while the majority of prokaryotic, viral, and organellar genes are coding.[4] Right now, we have genome sequences from 50 different bacterial phyla and 11 different archaeal phyla. Second-generation sequencing has yielded many draft genomes (close to 90% of bacterial genomes in GenBank are currently not complete); third-generation sequencing might eventually yield a complete genome in a few hours. The genome sequences reveal much diversity in bacteria. Analysis of over 2000 Escherichia coli genomes reveals an E. coli core genome of about 3100 gene families and a total of about 89,000 different gene families.[5] Genome sequences show that parasitic bacteria have 500-1200 genes, free-living bacteria have 1500-7500 genes, and archaea have 1500-2700 genes.[6] A striking discovery by Cole et al. described massive amounts of gene decay when comparing Leprosy bacillus to ancestral bacteria.[7] Studies have since shown that several bacteria have smaller genome sizes than their ancestors did.[8]Over the years, researchers have proposed several theories to explain the general trend of bacterial genome decay and the relatively small size of bacterial genomes. Compelling evidence indicates that the apparent degradation of bacterial genomes is owed to a deletional bias E. coli (fixed)DNA in nucleoid: blue cell membrane: green typical bacterial genome (eg. TB (M. tuberculosis) or E. coli ~4,000,000 bp ~1.3 mm of DNA ~1 Mbyte of information ~3,000 genes 4bp = 8bits = 1byte TGGC 11101001

bacterial genomes: information-rich, replicated very quickly

eg. Staphylococcus aureus (MRSA strain)~3 Mbp ~3,000 genes. all replicated in ~ 1 hour ( ! ) map shows ~ 0.6% of S. aureus genome ... ~ 18,000 bpproteins encoded in all 6 frames. but mostly on 1 strand (•• why?) all these 'files' (genes) need to be read (transcribed) most genes are 'on' by default ... eukaryotic genomes are even bigger ... bacterial genome (eg. M. tuberculosis) ~4,000,000 bp ~1 mm of DNA ~1 Mbyte ~3,000 genes human genome ~3,000,000,000 bp ~1 metre of DNA ~750 Mbytes ≤ 25,000 genes most genes 'off' by default digital devices store more information, take up far more space

DNA bending: essential to fit chromosomes into cells

fibroblasts ~2 m human genome fits into ~6 μm cell nucleus(wound on nucleosomes, + further condensation.. DNA structure on nucleosome core (eukaryotes only!) DNA bends fairly smoothly:~ 1.8 turns (700o) in ~ 140 bp DNA structure: B-like helix

Charge repulsion

force driving apart two ions, molecules, or regions of molecules of the same electric charge The DNA "backbone" has one negative charge on each phosphate group - so DNA is highly negatively charged overall in typical biological conditions! (at very low pH, the phosphates are protonated; that's whyit's called deoxyribonucleic acid. At typical cell pH, the negative charges are balanced by positive ions such as Na+ or Mg2+, diffusely in solution around the DNA molecule.) The negative charges repel each other, and the charged phosphates are hydrophilic; hence they'll prefer to be on the outside of the DNA structure

syn ( same ) and anti ( opposite)

syn and anti addition are different ways in which two substituents can be added to a double bond or triple bond. Syn addition is the addition of two substituentsto the same side (or face) of a double bond or triple bond, resulting in a decrease in bond order but an increase in number of substituents. In anti addition, two substituents are added to opposite sides (or faces) of a double bond or triple bond, once again resulting in a decrease in bond order and increase in number of substituents

DNA bending

the Crick and Watson model for DNA helix was correct ( ! ! )• most of the DNA in cells is similar to B-form double helix 2 important exceptions in living cells: replication and transcription however • DNA sequences vary in details of their structure, and in their flexibility every base-pair step differs slightly from regular B-DNA eg. in twist and roll angles. every DNA sequence has a unique structural 'character'

The Structure of Double-Stranded DNA

watson and Crick first described the structure of the DNA double helix in 1953. A representation of their model is shown in Figure 1.1. Duplex DNA is a right-handed helix formed by two individual DNA strands aligned in an antiparallel fashion. This means that one strand is oriented in the 5' ~3'direction and the other in the 3'~5' direction. The two strands are held to- gether by hydrogen bonds between individual bases. The bases are stackednear the center of the cylindrical helix. The base stacking provides consider- able stability to the double helix. The sugar and phosphate groups are on the outside of the helix and form a "backbone" for the helix. There are about 10 base pairs (bp) per turn of the double helix. Two important pieces of information were critical for the development of this structure, one of which was "Chargaff's Rules." In the early 1950s, Chargaff pointed out that the amount of adenine always equalled the amount of thymine and the amount of guanine always equalled the amount of cyto- sine (Chargaff, 1951; Zamenhof et al., 1952). This was true for DNA puri- fied from a wide variety of organisms, and true regardless of the total G + C (or A + T) content. This condition is met by having two strands of DNA in which the bases are hydrogen bonded with strict complementary base pair- ing. Specifically, A only pairs with T (A·T) and G only pairs with C (G· C). (See Table 1.1 for conventions in designating polynucleotide chains and base pairs.) The second piece of information came from X-ray diffraction patterns of DNA fibers (Wilkins and Randall, 1953; Wilkins et al., 1953) which showed that the geometric shape of DNA is a right-handed helix. Watson and Crick used this information to deduce a model for the structure of DNA. The double-helical model of DNA seems simple and straightforward. However, the elucidation of this structure was not trivial. Watson and Crick had no way of knowing that DNA was necessarily composed of two strands, that these strands were antiparallel, or that the bases were paired exclusively A . T and G· C. In addition, because of tautomerization and ionization, Watson and Crick were not even sure of the chemical form of the bases. This is a very important point because tautomerization and ionization change the electronic configuration of the bases, which changes their base-pairing prop- erties. Moreover, as discussed subsequently, there are many different ways to form hydrogen bonds between two bases

DNA bending plays an important role

~ DNA condensation and storage: nucleosome positions, etc.~ supercoiling: DNA circles (bacteria), DNA on nucleosomes (eukaryotes) ~ recognition of DNA sequences by proteins ( eg. IHF )