Biochemistry Test 2

Guanine pKa at n7

2.4

All DNA synthesis requires...

All DNA synthesis requires an RNA primer.

DNA gyrase

DNA gyrase is a bacterial topoisomerase that introduces negative supercoils into DNA. class II DNA gyrase (class II topoisomerase) catalyzes reaction involving relaxed circular DNA: • creates a nick in relaxed circular DNA • a slight unwinding at the point of the nick introduces supercoiling • the nick is resealed • The energy required for this process is supplied by the hydrolysis of ATP to ADP and Pi

What is the importance of pyrophosphatase in the synthesis of nucleic acids?

Hydrolysis of the pyrophosphate product prevents the reversal of the reac- tion by removing a product.

b-dna

It is thought to be the principal form that occurs in nature. right handed Base stacking bases are hydrophobic and interact by hydrophobic interactions in standard B-DNA 10 base pairs per turn (34Å) of the helix

Define replication, transcription, and translation.

Replication is the production of new DNA from a DNA template. Transcription is the production of RNA from a DNA template. Translation is the synthesis of proteins directed by mRNA, which reflects the base sequence of DNA.

Suggest a reason why it would be unlikely for rep- lication to take place without unwinding the DNA helix.

Replication requires separating the strands of DNA. This cannot happen unless the DNA is unwound.

Why is it necessary to unwind the DNA helix in the replication process?

Separating the two strands of DNA requires unwinding the helix.

Z-DNA

left handed Z-DNA is known to occur in nature, most often when there is a sequence of alternating purine- pyrimidine may play a role in gene expression The Z form of DNA can be considered a derivative of the B form of DNA, produced by flipping one side of the backbone 180° without having to break either the backbone or the hydrogen bonding of the complementary bases.

Processitivity

the number of nucleotides joined before the enzyme dissociates from the template The overall efficiency of DNA synthesis increases when the processivity of a polymerase increases.

why is DNA so stable?

the stacking of the bases in the native conformation of DNA contributes the largest part of the stabilization energy. Energy must be added to a sample of DNA to break the hydrogen bonds and to disrupt the stacking interactions. This is usually carried out by heating the DNA in solution.

Problems that need to be solved

• DNA needs to unwind • Strand separation introduces positive supercoils Polymerase must be loaded onto DNA Single strand needs to be protected from enzymatic hydrolysis Primer must be made and removed later on Okazaki fragments need to be linked

Eukaryotic DNA replication

*Even though many of the principles are the same, eukaryotic replication is more complicated in three basic ways: there are multiple origins of replication (replicators), the timing must be controlled to that of cell divisions, and more proteins and enzymes are involved. * The zones where replication is proceeding are called replicons, and the size of these varies with the species. In higher mammals, replicons may span 500 to 50,000 base pairs. Replication is initiated by a multisubunit protein called the origin recognition complex (ORC), which binds to the origin of replication. This protein complex appears to be bound to the DNA throughout the cell cycle, but it serves as an attachment site for several proteins that help control replication. The next protein to bind is an activation factor called the replication activator protein (RAP). After the activator protein is bound, replication licensing factors (RLFs) can bind. Yeast contains at least six different RLFs. They get their name from the fact that replication cannot proceed until they are bound. One of the keys to linking replication to cell division is that some of the RLF proteins have been found to be cytosolic. Thus, they have access to the chromosome only when the nuclear membrane dissolves during mitosis. Until they are bound, replication cannot occur. After RLFs bind, the DNA is then competent for replication. The combination of the DNA, ORC, RAP, and RLFs constitutes what researchers call the pre-replication complex (pre-RC). When these cyclins combine with CDKs, they can activate DNA replication and also block reassembly of a pre-RC after initia- tion. The state of activity of the CDKs and the cyclins determines the window of opportunity for DNA synthesis.

Fundamental Rules of DNA Replication

1. DNA replication is semiconservative (proven by Meselson-Stahl experiment) 2. Replication begins at an specific site (origin of replication) and is bidirectional.

adenine pKa at N1

3.8

cytosine pKa of N3

4.5

how many base pair turns in B-DNA?

8 turns ×10.5 bp/turn = 84 bp

thymine pKa of N3

9.5

Tm trends for DNA

A G-C base pair has three hydrogen bonds, and an A-T base pair has only two. The higher the percentage of G-C base pairs, the higher the melting temperature of a DNA molecule. G-C pairs are more hydrophobic than A-T pairs, so they stack better, which also affects the melting curve.

Base-Excision repair

A base that has been damaged by oxidation or chemical modification is removed by *DNA glycosylase*, leaving an AP site, so called because it is apurinic or apy- rimidinic (without purine or pyrimidine). An *AP endonuclease* then removes the sugar and phosphate from the nucleotide. An excision exonuclease then removes several more bases. Finally, DNA polymerase I fills in the gap, and DNA ligase seals the phosphodiester backbone. DNA glycosylases - enzymes that cleave the glycosidic bond in DNA Common DNA glycosylases: Uracil glycosylase Hypoxanthine glycosylase 3-Methyladenine glycosylase 7-Methyladenine glycosylase Others exist DNA repair is not made by inserting a new base reforming the glycosidic bond. Step 1. DNA glycosylase removes the damaged base creating an apurinic or apyrimidinic site (AP site). Step 2. An AP endonuclease recognizes the AP site, cleaves the sugar phosphate backbone, and generates a 3'-OH or 5'-phosphate (varies depends on the AP nucleosidase). Step 3. pol I removes the deoxyribose phosphate and a segment of DNA. The resulting gap is then "filled in" using pol I. In eukaryotes, specialized repair DNA polymerases catalyze this step. Step 4. DNA ligase forms a new phosphodiester bond linking together the newly synthesized (repaired) DNA and the "old" DNA.

nucleoside

A nucleoside is a compound that consists of a base and a sugar covalently linked together. It differs from a nucleotide by lacking a phosphate group in its structure. In a nucleoside, a base forms a glycosidic linkage with the sugar. When the sugar is β-D-ribose, the resulting compound is a *ribonucleo- side*; when the sugar is β-D-deoxyribose, the resulting compound is a *deoxyri- bonucleoside* The glycosidic linkage is from the C-1' carbon of the sugar to the N-1 nitrogen of pyrimidines or to the N-9 nitrogen of purines. • This bond is quite stable toward hydrolysis, esp. in pyrimidines • Bond cleavage is catalyzed by acid

sugar-phosphate backbone

A portion of a DNA chain differs from the RNA chain just described only in the fact that the sugar is 2'-deoxyribose rather than ribose (Figure 9.6). In abbreviated notation, the deoxyribonucleotide is specified in the usual man- ner. Sometimes a letter d is added to indicate a deoxyribonucleotide residue; for example, dG is substituted for G, and the deoxy analogue of the ribooligo- nucleotide in the preceding paragraph would be d(GACAT). However, given that the sequence must refer to DNA because of the presence of thymine, the sequence GACAT is not ambiguous and would also be a suitable abbreviation. The inside diameter of the sugar-phosphate backbone of the double helix is about 11 Å

What is a replication fork? Why is it important in replication?

A replication fork is the site of formation of new DNA. The two strands of the original DNA separate, and a new strand is formed on each original strand.

Semidiscontinuous DNA Replication

All synthesis of nucleotide chains occurs in the 5' 3 3' direction from the perspective of the chain being synthesized. This is due to the nature of the reaction of DNA synthesis. The last nucleotide added to a growing chain has a 3'-hydroxyl on the sugar. The incoming nucleotide has a 5'-triphosphate on its sugar. The 3'-hydroxyl group at the end of the growing chain is a nucleophile. It attacks the phosphorus adjacent to the sugar in the nucleotide to be added to the growing chain, leading to the elimination of the pyrophosphate and the formation of a new phosphodiester bond Because both strands are synthesized in concert by a dimeric DNA polymerase situated at the replication fork, the 5 --> 3 parental strand must wrap around in trombone fashion so that the unit of the dimeric DNA polymerase replicating it can move along it in the 3 --> 5 direction. This parental strand is copied in a discontinuous fashion because the DNA polymerase must occasionally dissociate 3 from this strand and rejoin it further along. The Okazaki fragments 5 are then covalently joined by DNA ligase to form an uninterrupted DNA strand.

DNA gyrase

An enzyme called DNA gyrase (class II topoisomerase) catalyzes the conversion of relaxed, circular DNA with a nick in one strand to the supercoiled form with the nick sealed that is found in normal prokaryotic DNA The energy required for the process is supplied by the hydrolysis of ATP. Prokaryotic DNA is negatively supercoiled and opening of the helix during replication introduces positive supercoils. DNA gyrase fights these positive supercoils by putting negative supercoils ahead of the replication fork

Describe the structural features of an origin of replication.

An origin of replication consists of a bubble in the DNA. There are two places at opposite ends where new polynucleotide chains are formed

How is single-stranded DNA protected long enough for replication?

Another protein, called the *single-strand binding protein* (SSB), stabilizes the single-stranded regions by binding tightly to these portions of the molecule. The presence of this DNA-binding protein protects the single-stranded regions from hydrolysis by the nucleases.

binding shit to DNA

At neutral, physiological pH, each phosphate group of the backbone carries a negative charge. Positively charged ions, such as Na+ or Mg2+, and polypeptides with positively charged side chains must be associated with DNA in order to neutralize the negative charges. Eukaryotic DNA, for example, is complexed with histones, which are positively charged proteins, in the cell nucleus.

double helix more info

Base pairs other than A-T and G-C are possible, but they do not have the correct hydrogen bonding pattern (A-C or G-T pairs) or the right dimensions (purine-purine or pyrimidine-pyrimidine pairs) to allow for a smooth double helix The outside diameter of the helix is 20 Å (2 nm). The length of one complete turn of the helix along its axis is 34 Å (3.4 nm) and contains 10 base pairs.

base stacking

Base-stacking interactions increase with increasing salt concentration, as high salt concentrations mask the destabilising charge repulsion between the two negatively charged phosphodiester backbones. DNA double strand stability therefore increases with increasing salt concentration. Divalent cations such as Mg2+ are more stabilising than Na+ ions, and some metal ions bind to specific loci on the DNA duplex.

How are DNA lesions produced?

Chemistry of the nucleotides Tautomer formation (discussed) Non-enzymatic reactions Environmental Damage *Deamination or loss of exocyclic amino groups* Hydrolysis of the glycosidic Bond Slow Rate, ~10-5 day DNA only, hydrolysis of the glycosidic bond is much slower More common for purines than pyrimidines Loss of: purine results in an apurinic site pyrimidine results in an apyrimidinic site

topoisomerase

Class I topoisomerases cut the phosphodiester backbone of one strand of DNA, pass the other end through, and then reseal the backbone. Class II topoisomerases cut both strands of DNA, pass some of the remaining DNA helix between the cut ends, and then reseal.

Different types of polymerase

DNA polymerase I (Pol I) was discovered first, with the subsequent discovery of polymerases II (Pol II) and polymerase III (Pol III). Polymerase I consists of a single polypeptide chain, but polymerases II and III are multisubunit proteins that share some common subunits. Polymerase II is not required for replication; rather, it is strictly a repair enzyme. Recently, two more polymerases, Pol IV and Pol V, were discovered. They, too, are repair enzymes, and both are involved in a unique repair mechanism called the SOS response

Compare and contrast the properties of the enzymes DNA polymerase I and polymerase III from E. coli.

DNA polymerase I is primarily a repair enzyme. DNA polymerase III is mainly responsible for the synthesis of new DNA

DNA polymerase

DNA polymerase catalyzes the successive addition of each new nucleotide to the growing chain.

Pol I and II

DNA polymerases I and II are involved in proofreading and repair processes.

Why is it more important for DNA to be replicated accurately than transcribed accurately?

DNA represents the permanent copy of genetic information, whereas RNA is transient. The cell could survive production of some mutant proteins, but not DNA mutation.

Origin of replication

During replication, the DNA double helix unwinds at a specific point called the origin of replication Two possibilities exist for the growth of the new strands: synthesis can take place in both directions from the origin of replication, or in one direction only. It has been established that DNA synthesis is *bidirectional in most organisms, with the exception of a few viruses and plasmids.*

nucleosome

Each "bead" is a nucleosome, consisting of DNA wrapped around a histone core. This protein core is an octamer, which includes two molecules of each type of histone but H1; the composition of the octamer is (H2A)2(H2B)2(H3)2(H4)2. The "string" portions are called spacer regions; they consist of DNA complexed to some H1 histone and nonhistone proteins. As the DNA coils around the histones in the nucleosome, about 150 base pairs are in contact with the proteins; the spacer region is about 30 to 50 base pairs long. Histones can be modified by acetylation, methylation, phosphorylation, and ubiquitinylation. Ubiquitin is a protein involved in the degradation of other proteins. each bead is a nucleosome Nucleosome consists of: DNA wrapped around histone core Nucleosome core is octamer: (H2A)2(H2B) 2(H3) 2(H4) 2 H1 histone is associated with the "string" portion between the beads (the spacer region)

Semiconservative replication

Each new DNA molecule contains one strand from the original DNA and one newly synthesized strand Semiconservative replication of DNA was established unequivocally in the late 1950s by experiments performed by Matthew Meselson and Franklin Stahl. E. coli bacteria were grown with 15NH4Cl as the sole nitrogen source, 15N being a heavy isotope of nitrogen. (The usual isotope of nitrogen is 14N.) In such a medium, all newly formed nitrogen compounds, including purine and pyrimidine become nucleobases, labeled with 15N.

How does supercoiling take place in eukaryotic DNA?

Electrostatic attraction between the negatively charged phosphate groups on the DNA and the positively charged groups on the proteins favors the formation of complexes of this sort. The resulting material is called chromatin. Thus, topological changes induced by supercoiling must be accommodated by the histone-protein component of chromatin.

How does prokaryotic DNA supercoil into its tertiary structure?

Enzymes that affect the supercoiling of DNA have been isolated from a variety of organisms. Naturally occurring circular DNA is negatively supercoiled except during replication, when it becomes positively supercoiled.

Proofreading and repair

Errors in replication occur spontaneously only once in every 10^9 to 10^10 base pairs. *Proofreading* refers to the removal of incorrect nucleotides immediately after they are added to the growing DNA during the replication process. Pol I can be cleaved into two major fragments. One of them (the Klenow fragment) contains the polymerase activity and the proofreading activity. The other con- tains the 5' -> 3' repair activity. DNA polymerase I uses its 3' exonuclease activity to remove the incorrect nucleotide. (3' -> 5') Although the specificity of hydrogen-bonded base pairing accounts for one error in every 104 to 105 base pairs, the proofreading function of DNA polymerase improves the fidelity of replication to one error in every 10^9 to 10^10 base pairs. The "proof-reading" activity improves the accuracy of DNA polymerase by a factor of 102-103. All DNA polymerases have a 3'→ 5' exonuclease activity. The 3'→ 5' exonuclease activity is called the "proofreading activity" of DNA polymerase. This enables DNA polymerase to remove and replace incorrect bases. Overall error rate of DNA synthesis: 1 error per 10^9-10^10 nucleotides added DNA polymerase: Base-pairing interactions: 1 error per 10^4-10^5 nucleotides added "Proof-reading" activity: decrease in error 10^2-10^3- fold. SUM: 1 error per 10^6-10^8 nucleotides added

Replication forks

For each origin of replication, there are two points *(replication forks)* at which new polynucleotide chains are formed.

Use of a primer

If DNA polymerases are added to a single-stranded DNA template with all the deoxynucleotide triphosphates necessary to make a strand of DNA, no reaction occurs. It was discovered that DNA polymerases cannot catalyze de novo synthesis. All three enzymes require the presence of a primer, a short oligonucleotide strand to which the growing polynucleotide chain is covalently attached in the early stages of replication. In essence, DNA polymerases must have a nucleotide with a free 3'-hydroxyl already in place so that they can add the first nucleotide as part of the growing chain. In natural replication, this primer is RNA.

How do DNA and RNA differ?

Important differences between DNA and RNA appear in their secondary and tertiary structures

The Flow of Genetic Information in the Cell

In nearly all organisms, the flow of genetic information is DNA -> RNA -> protein. The only major exceptions are some viruses (called retroviruses) in which RNA, rather than DNA, is the genetic material. In those viruses, RNA can direct its own synthesis as well as that of DNA. The enzyme reverse transcriptase catalyzes this process. ■ Before cells divide, they must synthesize a new copy of DNA. This process is called replication. ■ When DNA is used as a template to synthesize RNA, the process is called transcription, and is the subject of the next chapter. ■ The RNA sequence of messenger RNA is used to direct the synthesis of proteins in a process called translation.

density-gradient centrifugation

In this experiment, the 15N-labeled cells were then transferred to a medium that contained only 14N. This technique depends on the fact that heavy 15N DNA (DNA that contains 15N alone) forms a band at the bottom of the tube; light 14N DNA (containing 14N alone) appears at the top of the tube. DNA containing a 50-50 mixture of 14N and 15N appears at a position halfway between the two bands. In the actual experiment, this 50-50 hybrid DNA was observed after one generation, a result to be expected with semiconservative replication. After two generations in the lighter medium, half of the DNA in the cells should be the 50-50 hybrid and half should be the lighter 14N DNA.

Do DNA-polymerase enzymes also function as exonucleases?

Most DNA-polymerase enzymes also have exonuclease activity.

Replication of DNA -> 3 steps

Naturally occurring DNA exists in many forms. Single- and double-stranded DNAs are known, and both can exist in linear and circular forms. The cell faces three important challenges in carrying out the necessary steps. The first challenge is separating the two DNA strands. The two strands of DNA are wound around each other in such a way that they must be unwound if they are to be separated. In addition to achieving continuous unwinding of the double helix, the cell also must protect the unwound portions of DNA from the action of nucleases that preferentially attack single-stranded DNA. The second task involves synthesizing of DNA from the 5' to the 3' end. Two antiparallel strands must be synthesized in the same direction on antiparallel templates. In other words, the template has one 5' 3 3' strand and one 3' 3 5' strand, as does the newly synthesized DNA. The third task is guarding against errors in replication, ensuring that the correct base is added to the growing poly- nucleotide chain.

How can replication proceed along the DNA if the two strands are going in opposite directions?

One newly formed strand (the leading strand) is formed continuously from its 5' end to its 3' end at the replication fork on the exposed 3' to 5' template strand. The other strand (the lagging strand) is formed semidiscontinuously in small fragments (typically 1000 to 2000 nucleotides long), sometimes called Okazaki fragments after the scientist who first studied them (Figure 10.6). The 5' end of each of these fragments is closer to the replication fork than the 3' end. The fragments of the lagging strand are then linked together by an enzyme called DNA ligase. • Synthesis always occurs by addition of new nucleotides to the 3' end. • The leading strand is made continuously as the replication fork advances. • The lagging strand is made discontinuously in short pieces (Okazaki fragments) that are then joined. • Okazaki fragments vary in length depending on cell type. Prokaroytes: 1000-2000 nucleotides Eukaryotes: 100-200 nucleotides

Why does DNA contain thymine instead of uracil?

One of the most common spontaneous mutations of bases is the natural deamination of cytosine. At any moment, a small but finite number of cytosines lose their amino groups to become uracil. Imagine that during repli- cation, a C-G base pair separates. If at that moment the C deami- nates to U, it would tend to base-pair to A instead of to G. If U were a natural base in DNA, the DNA polymerases would just line up an adenine across from the uracil, and there would be no way to know that the uracil was a mistake. This would lead to a much higher level of mutation during replication. Because uracil is an unnatural base in DNA, DNA polymerases can recognize it as a mistake and can replace it. Thus, the incorporation of thymine into DNA, though energetically more costly, helps ensure that the DNA is replicated faithfully.

DNA synthesis always takes place from the 5' to the 3' end. The template strands have opposite directions. How does nature deal with this situation?

One strand of newly formed DNA uses the 3′-to-5′ strand as a template. The problem arises with the 5′-to-3′ strand. Nature deals with this issue by using short stretches of this strand for a number of chunks of newly formed DNA. They are then linked by DNA ligase

Bidirectional replication

One such bubble (and one origin of replication) exists in the circular DNA of prokaryotes In eukaryotes, several origins of replication, and thus several bubbles, exist The bubbles grow larger and even- tually merge, giving rise to two complete daughter DNAs. This bidirectional growth of both new polynucleotide chains represents net chain growth. *Both new polynucleotide chains are synthesized in the 5' to 3' direction.*

Pol III

Pol III is the principal enzyme responsible for synthesis of new DNA, and it is a multiple-subunit enzyme.

Eukaryotic DNA polymerases

Polymerase α was the first discovered, and it has the most subunits. It also has the ability to make primers, but it lacks a 3' 3 5' proofreading activity and has low processivity. Polymerase δ is the principal DNA polymerase in eukaryotes. DNA polymerase β appears to be a repair enzyme. DNA polymerase γ carries out DNA replication in mitochondria.

Differences in DNA Replication in Prokyryotes and Eukaryotes

Prokaryotes Five polymerases (I, II, III, IV, V) Functions of polymerase: I is involved in synthesis, proofreading, repair, and removal of RNA primers II is also a repair enzyme III is main polymerizing enzyme IV, V are repair enzymes under unusual conditions Polymerases are also exonucleases One origin of replication Okazaki fragments 1000-2000 residues long No proteins complexed to DNA Eukaryotes: Five polymerases (α, β, γ, δ, ε) Functions of polymerases: α: a polymerizing enzyme β: is a repair enzyme γ: mitochondrial DNA synthesis δ: main polymerizing enzyme ε: function unknown Not all polymerases are exonucleases Several origins of replication Okazaki fragments 150-200 residues Section 10.6 Summary long Histones complexed to DNA

major types of RNA

Six kinds of RNA—transfer RNA (tRNA), ribosomal RNA (rRNA), messenger RNA (mRNA), small nuclear RNA (snRNA), micro RNA (miRNA), and small interfering RNA (siRNA)—play an important role in the life processes of cells. The base sequences of all types of RNA are determined by that of DNA. The process by which the order of bases is passed from DNA to RNA is called transcription

Different types of mutations

Substitution mutation: replacement of one base with another Insertion or Deletion mutation: the addition or loss of one or more bases to the DNA Silent mutation: one that has no effect on gene function

Telomerase and cancer

Telomeres: This repetitive DNA is noncoding and acts as a buffer against degradation of the DNA sequence at the ends, which would occur with each replication as the RNA primers are degraded. Telomerase: a ribonuclear protein, containing a section of RNA that is the complement of the telomere. In humans, this sequence is 5'CCCUAA3'. Telomerase binds to the 5' strand at the chromo- some end and uses a reverse transcriptase activity to synthesize DNA (shown in red) on the 3' strand, using its own RNA as the template. This allows the template strand (shown in purple) to be elongated, effectively lengthening the telomere.

proofreading and repair

The 3' -> 5' exonuclease activity, which all three polymerases possess, is part of the *proofreading* function; incorrect nucleotides are removed in the course of replication and are replaced by the correct ones. Proofreading is done one nucleotide at a time. The 5' -> 3' exonuclease activity clears away short stretches of nucleotides during *repair*, usually involving several nucleotides at a time. This is also how the RNA primers are removed. The proofreading-and-repair function is less effective in some DNA polymerases.

Replisome

The entire complex, including the DNA polymerases, is called the replisome.

How can we monitor DNA denaturation?

The heat denaturation of DNA, also called melting, can be monitored experimentally by observing the absorption of ultraviolet light. The bases absorb light in the *260-nm-wavelength* region. It is based on the fact that the bases, which are stacked on top of one another in native DNA, become unstacked as the DNA is denatured. As the DNA is heated and the strands separate, the wavelength of absorption does not change, *but the amount of light absorbed increases* -> hyperchromacity Because the bases interact differently in the stacked and unstacked orienta- tions, their absorbance changes

Holliday model

The model for how recombination occurs between *homologous chromosomes*

Comment on the dual role of the monomeric reactants in replication.

The monomeric reactants are dNTP (deoxynucleotidetriphosphates). These will be cleaved into dNMP, and the dNMPs will be incorporated into the DNA. Cleavage of the dNTP phosphoester bond also releases pyrophosphate and the energy that "powers" replication. This energy is rather large (question 10.17): this "overkill" ensures that the reaction is only in 1 direction

How do nucleotides combine to give nucleic acids?

The polymerization of nucleotides gives rise to nucleic acids. The linkage between monomers in nucleic acids involves formation of two ester bonds by phosphoric acid. The hydroxyl groups to which the phosphoric acid is esterified are those bonded to the 3' and 5' carbons on adjacent residues. The resulting repeated linkage is a 3,'5'-phosphodiester bond.

Primosome

The primer and the protein molecules at the replication fork constitute the primosome.

histones

The principal proteins in chromatin are the histones, of which there are five main types, called H1, H2A, H2B, H3, and H4. All these proteins contain large numbers of basic amino acid residues, such as lysine and arginine. the DNA is tightly bound to all the types of histone except H1. The H1 protein is comparatively easy to remove from chromatin, but dis- sociating the other histones from the complex is more difficult.

Define processivity, and indicate the importance of this concept in DNA replication.

The processivity of a DNA polymerase is the number of nucleotides incor- porated before the enzyme dissociates from the template. The higher this number, the more efficient the replication process.

nucleophilic attack of DNA synthesis

The reaction of DNA synthesis involves the nucleophilic attack of the 3'- hydroxyl of one nucleotide on the γ-phosphate of the incoming nucleo- side triphosphate.

Why is the replication of DNA referred to as a semiconserva- tive process? What is the experimental evidence for the semiconser- vative nature of the process? What experimental results would you expect if replication of DNA were a conservative process?

The semiconservative replication of DNA means that a newly formed DNA molecule has one new strand and one strand from the original DNA. The experimental evidence for semiconservative replication comes from density-gradient centrifugation (Figure 10.3). If replication were a conserva- tive process, the original DNA would have two heavy strands and all newly formed DNA would have light strands.

Synthesis and linking of new DNA strands

The synthesis of two new strands of DNA is begun by DNA polymerase III. The newly formed DNA is linked to the 3'-hydroxyl of the RNA primer, and synthesis proceeds from the 5' end to the 3' end on both the leading and the lagging strands. Two molecules of Pol III, one for the leading strand and one for the lagging strand, are physically linked to the primosome. The resulting multiprotein complex is called the replisome. As the replication fork moves, the RNA primer is removed by polymerase I, using its exonuclease activity. The primer is replaced by deoxynucleotides, also by DNA polymerase I, using its polymerase activity.

Nucleotide-excision repair

UV light; When a serious lesion, such as a pyrimidine dimer, is detected, ABC excinuclease binds to the region and cuts out a large piece of DNA, including the lesion. DNA polymerase I and DNA ligase then resynthesize and seal the DNA.

How does proofreading improve replication fidelity?

When C, a rare tautomeric form of cytosine (C**) pairs with A and is incorporated into the lengthening strand, the nucleotide is mismatched. The misfired 3'-OH end of the growing strand blocks further elongation. DNA polymerase slides back to position the misfired base in the 3'->5' exonuclease active site. The mispaired nucleotide is removed and DNA polymerase slides forward and resumes its polymerization activity. *nick translation*: DNA polymerase I is able to use its 5' -> 3' exonuclease activity to remove RNA primers or DNA mistakes as it moves along the DNA. It then fills in behind it with its polymerase activity. When organisms are exposed to *mutagens* like UV light (UV damage: thymine dimers), radioactivity, etc., may create *pyrimidine dimers* that distorts the normal shape of DNA and interferes with replication and transcription. Free radicals can also interrupt the phosphodiester backbone.

nucleoside -> nucleotide

When phosphoric acid is esterified to one of the hydroxyl groups of the sugar portion of a nucleoside, a nucleotide is formed The 5' nucleotides are most commonly encountered in nature.

What is the nature of the DNA double helix?

When the double helix was proposed by James Watson and Francis Crick in 1953, it touched off a flood of research activity, leading to great advances in molecular biology. Hydrogen bonds between bases on opposite chains determine the alignment of the helix, with the paired bases lying in planes perpendicular to the helix axis. The sugar- phosphate backbone is the outer part of the helix. The chains run in antiparallel directions, one 3' to 5' and the other 5' to 3.' The X-ray diffraction pattern of DNA demonstrated the helical structure and the diameter. The combination of evidence from X-ray diffraction and chemi- cal analysis led to the conclusion that the base pairing is complementary, meaning that adenine pairs with thymine and that guanine pairs with cytosine. Because complementary base pairing occurs along the entire double helix, the two chains are also referred to as complementary strands. *An adenine-thymine (A-T) base pair has two hydrogen bonds between the bases; a guanine-cytosine (G-C) base pair has three*

RNA primer

a short oligonucleotide strand to which the growing polynucleotide chain is covalently attached in the early stages of replication. The primer (RNA) is hydrogen-bonded to the template (DNA); the primer provides a stable framework on which the nascent chain can start to grow. The newly synthesized DNA strand begins to grow by forming a covalent linkage to the free 3'-hydroxyl group of the primer.

major groove

both can be sites at which drugs or polypeptides bind to DNA

denaturation of DNA can be induced by...

changes in pH and high temperature • Covalent bonds remain intact • Genetic code remains intact • Hydrogenbondsarebroken • Two strands separate • Base stacking is lost • UV absorbance increases Denaturation can be induced by high temperature, or change in pH . Denaturation may be reversible: annealing as strands separate, absorbance at 260 nm increases AT rich regions melt at a lower temperature than GC-rich regions

Mismatch repair

enzymes recognize that two bases are incorrectly paired. The area with the mismatch is removed, and DNA polymerases replicate the area again. If there is a mismatch, the challenge for the repair system is to know which of the two strands is the correct one. This is possible only because prokaryotes alter their DNA at certain locations (Chapter 13) by modifying bases with added methyl groups. This methylation occurs shortly after replication. Thus, immediately after replication, there is a window of opportunity for the mismatch-repair system. For example, Originally, both parental strands are methylated.Because the parental strand contained methylated adenines, the enzymes can distinguish the parental strand from the newly synthesized daughter strand without the modified bases. Thus, the T is the mistake and not the G. Several proteins and enzymes are then involved in the repair process. MutH, MutS, and MutL form a loop between the mistake and a methylation site. DNA helicase II helps unwind the DNA. Exonuclease I removes the section of DNA containing the mistake. Single-stranded binding proteins protect the template (blue) strand from degradation. DNA polymerase III then fills in the missing piece • Mismatch repair replaces bases normally found in DNA and, therefore, are not recognized by the direct repair and excision repair mechanisms. • Eight possible mismatches: A-C, A-G, A-A, C-C, C-T, T-T, T-G, and G-G. • The 8 mismatches do not occur with equal frequency and are not repaired with equal efficiency. Immediately after replication, there is a short lag during which the template strand is methylated, but the newly synthesized strand is not. This is called hemi- methylated DNA. Transient undermethylation of the daughter stand is the chemical basis of strand discrimination. The methylated strand serves as the template for repair. • The methylation sites (GATC) are usually separated by ~2000 bp. • Thus, the site of the mismatch may be as far as ~1,000 bp from the nearest methylated site. • Mismatches are nearly always corrected to correspond to the information in the template strand. What does this mean? The mismatch repair system "assumes" that the template strand contains the correct base. • This means that E. coli must discriminate between the template and newly synthesized strands. • Strand discrimination is accomplished by modifying the template strand with methyl groups. • The enzyme Dam methylase methylates the N6-position of the adenine within the (5')GATC sequence.

a-dna

has 11 base pairs for each turn of the helix. Its base pairs are not perpendicular to the helix axis but lie at an angle of about 20° to the perpendicular right handed occurs at high salt conditions

monomers of nucleic acids

nucleotides An individual nucleotide consists of three parts—a nitrogenous base, a sugar, and a phosphoric acid residue—all of which are covalently bonded together. The order of bases in the nucleic acids of DNA contains the information necessary to produce the correct amino acid sequence in the cell's proteins.

nucleic acid bases

pyrimidines: single ring aromatic compounds *Cytosine, thymine, and uracil* All are good H-bond donors and acceptors The common *purine bases* (double-ring aromatic compounds) are adenine and guanine, both of which are found in RNA and in DNA (Figure 9.1). *good H-bond donors and acceptors* In addition to these five commonly occurring bases, there are "unusual" bases, with slightly different structures, that are found principally, but not exclusively, in transfer RNA

Primase

responsible for copying a short stretch of the DNA template strand to produce the RNA primer sequence.

micro RNA

small affects gene expression; growth and development

small interfering RNA

small affects gene expression; used by scientists to knock out a gene being studied

small nuclear RNA

small processes initial mRNA to its mature form in eukaryotes

tRNA

small transports amino to site of protein synthesis • the smallest kind of the three RNAs • a single-stranded polynucleotide chain between 73-94 nucleotide residues • carries an amino acid at its 3' end • intramolecular hydrogen bonding occurs in tRNA • modified bases occur in tRNA -A form DNA helix The hydrogen-bonded portions of the molecule are called stems, and the non-hydrogen-bonded portions are loops. A particular tertiary structure is necessary for tRNA to interact with the enzyme that covalently attaches the amino acid to the 2' or 3' end. To produce this tertiary structure, the tRNA folds into an L-shaped conformation that has been determined by X-ray diffraction

mRNA

variable directs amino acid sequence of proteins a ribonucleic acid that carries coded genetic information from DNA to ribosomes for the synthesis of proteins • present in cells in relatively small amounts and very short-lived • single stranded • biosynthesis is directed by information encoded on DNA • a complementary strand of mRNA is synthesized along one strand of an unwound DNA, starting from the 3' end • little (no) secondary structure

rRNA

variable in size combines with proteins to form ribosomes the site of protein synthesis

DNA recombination

• DNA recombination is a natural process in which genetic information is rearranged to form new associations. • If the recombination involves a reaction between homologous sequences, then the process is called homologous recombination. When very different nucleotide sequences recombine, it is non-homologous recombination. • Recombination *does not* occur randomly around a chromosome. There are some areas of a chromosome, called hot spots, that are much more likely to show recombination. • Recombination occurs by the breakage and reunion of DNA strands so that physical exchange of DNA parts takes place. The mechanism was deduced in 1964 by Robin Holliday and is referred to as the Holliday Model

Information Flow within the Cell

• Information from parental DNA is copied to daughter DNA with high fidelity via DNA replication • RNA is synthesized using DNA as a template during transcription • Viruses are able to make RNA and DNA using RNA as a template in reverse transcription • Proteins are synthesized based on the information stored in ribonucleotide triplets in RNA • Information encoded in the nucleotide sequence of DNA is transcribed through RNA synthesis • Sequence then dictated by DNA sequence • Central dogma of molecular biology • In addition to their roles as the monomeric units of the DNA and RNA, the nucleotides serve other KEY biological functions! - Transfer of energy: ATP, GTP, and the other 5'- nucleotide triphosphates. - Signal transduction: cAMP and cGMP - Enzyme cofactors: Coenzyme A - acyl group transfer NAD+/NADH and NADP+/NADPH - redox chemistry • Strand separation occurs first • Each strand serves as a template for the synthesis of a new strand • Synthesis is catalyzed by enzymes known as DNA polymerases • Newly made DNA molecule has one daughter strand and one parent strand.

The Primase Reaction

• The primase reaction • RNA serves as a primer in DNA replication • primer activity first observed in-vivo. • Primase - catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence

DNA pol has help!

• Topoisomerases: remove and introduce supercoils • Helicase: a helix-destabilizing protein, promotes unwinding by binding at the replication fork • Single-stranded binding (SSB) protein: stabilizes single-stranded regions by binding tightly to them. • Primase: synthesis of RNA primers • DNA ligase: covalently link together Okazaki fragments

Function of DNA polymerase

• all four deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP • Mg2+ • an RNA primer - a short strand of RNA to which the growing polynucleotide chain is covalently bonded in the early stages of replication (in a test-tube, ssDNA can act as a primer as well) • DNA-Pol I: repair &patching of DNA; primer removal • DNA-Pol III: responsible for the polymerization of the newly formed DNA strand • DNA-Pol II, IV, and V: proofreading and repair enzymes