Lecture 6

Pyrimidines (Thymine, Cytosine, Uracil)

in DNA, the pyrimidines are cytosine and thymine; in RNA, the pyrimidines are cytosine and uracil. One result is that pyrimidines are planar molecules, and purines are very nearly planar, with just a slight pucker. Pyrimidines are generally restricted to the anti conformation, because of steric in- terference between the sugar and the carbonyl oxygen at C-2 of the pyrimidine.

Chargaff (Watson and Crick)

In all cellular DNAs, regardless of the species, the number of adenosine residues equals the number of thymidine residues (i.e., A = T), and the number of guanosine residues equals the number of cytidine residues (G = C). From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine residues: A+G=T+C. Chargaff's discoveries imposed important constraints on possible models for the structure of DNA. The challenge then was to formulate a three-dimensional model of the DNA molecule that could account not only for the x-ray diffraction data but also for the specific A = T and G = C base equivalences discovered by Chargaff.

Cruciform and hairpin (in vitro)

Inverted repeats within a single strand of DNA can be converted into a hairpin, which is double-stranded in the stem region. Inverted repeats within a double-stranded DNA sequence can form a cruciform (double- hairpin) structure. In-vivo these are linked with genomic instability

Cytidine

Name given for just the cystine nitrogen base

D-deoxyribose

Nature uses only the D-enantiomeric form of the sugars—D-ribose or D-deoxyribose—as the building blocks for DNA and RNA. the pentose is 2'-deoxyribose, which has no hydroxyl group on the 2' carbon ( Only exists in the closed ring form The pentose ring of each deoxyribose is in the C-2' endo conformation (see Figure 6-3), and this sugar pucker defines the distance between adjacent phosphate groups in the DNA backbone.

Triple or quadruple helices (in vitro)

One layer of a guanosine tetraplex structure, showing hydrogen bonding of the bases. A K􏰄 ion in the center of the tetraplex stabilizes the structure by coordinating the bases' functional groups. Detected only in vitro

Double Helix DNA (dsDNA)

When a double-stranded sequence is shown, the top strand is written in the 5'→3' direction. The most important hydrogen- bonding patterns are those defined by Watson and Crick in 1953, in which A hydrogen-bonds specifically with T (or U), and G with C (Figure 6-11 on page 184). These two types of base pairs predominate in double- stranded DNA and RNA (and thus are considered the canonical base pairs). The stability of the DNA double helix arises primarily from the hydrophobic base-stacking inter- actions, which are largely nonspecific with respect to sequence. Fur- thermore, extensive networks of weak bonds in double- stranded DNA, such as van der Waals interactions and hydrogen bonds, are arranged so that for most of these bonds, they cannot break without simultaneously break- ing many others. By far the most significant property of the double helix as an information carrier is the hydrogen bond- ing between the bases.

ssDNA

When a ssDNA sequence is given, it is written and read in the direction going from the free 5'-P to the free 3'-OH, left to right

Z-DNA

Z-DNA, which forms only under high salt conditions or with C   G-rich DNA sequences, is left- handed, and its backbone has a zigzag pattern. Z-DNA forms a left-handed helix that contains 12 base pairs per turn and occurs only in sequences rich in C and G residues.

DNA nanotechnology

aims to make intelligent structures

C-2' endo form (deoxyribose)

4 of the atoms of the ring lay in one plane The fifth (either C-2' or C-3') is out of the plane The pentose ring of each deoxyribose is in the C-2' endo conformation (see Figure 6-3), and this sugar pucker defines the distance between adjacent phosphate groups in the DNA backbone. the sugar pucker of the pentose (C-2' endo vs. C-3' endo). Has Important consequences on the structure of the double helix (defines the distance between adjacent phosphate groups in the DNA backbone. )

Nucleotide

A nucleotide is a molecule consisting of three charac- teristic components: a heterocyclic base, a five-carbon sugar called a pentose, and a phosphate group. Because of their different pentose components, the structural units of DNAs and RNAs are known as deoxyribonucleotides (deoxyribonucleoside 5'- monophosphates) and ribonucleotides The successive nucleotides of DNA and RNA are cova- lently joined through phosphate group "connectors" in which the 5'-phosphate group of one nucleotide unit is linked to the 3'-hydroxyl group of the next nucleotide, creating a phosphodiester bond Polynucleotides (nucleic acids) have a directionality defined by a 5' terminus and a 3' terminus. OH denotes a 3'-hydroxyl group.

oligonucelotides (synthetic synthesis of DNA)

A short nucleic acid containing 50 or fewer nucleo- tides is generally called an oligonucleotide; a longer nucleic acid is a polynucleotide. The oligonucleotide is synthesized in the 3' → 5' direction, starting with a single nucleotide that is covalently attached to a solid support, such as a glass bead.

A-DNA

A-DNA is favored in many solutions that are rela- tively devoid of water. In this case, the DNA is still arranged in a right-handed double helix, but the helix is wider and the number of base pairs per helical turn is 11, rather than 10.5 as in B-DNA. the base pairs in A-DNA are tilted above the plane by about +20*. In the C-3' endo form. Like B-DNA, A-DNA is right-handed, but it is more compact, with 11 base pairs per turn.

deoxyribonucleotides

Because of their different pentose components, the structural units of DNAs and RNAs are known as deoxyribonucleotides (deoxyribonucleoside 5'- monophosphates) and ribonucleotides

Epigenetic Modifications (DNA)

Certain nucleotide bases in DNA molecules are en- zymatically methylated, usually after DNA synthe- sis is complete. Adenine and cytosine are methyl- ated more frequently than guanine. More than half of all CpG sequences in mammalian genomes are methylated on the C residue

Sugars (DNA and RNA)

RNA is chemically distinct from DNA, because it contains a different kind of sugar in its nucleotide building blocks. They both are a five-carbon sugar called a pentose. In DNA, the pentose is 2'-deoxyribose, which has no hydroxyl group on the 2' carbon (red); in RNA, the sugar is ribose, which includes a 2' hydroxyl. The predominant type of sugar pucker that characterizes DNA differs from that found in RNA, re- sulting in the different shapes and geometries of the DNA and RNA double helices, as we'll see later in this chapter. The alternating phosphate and sugar residues form the backbone of the nucleic acid, and the bases can be viewed as side groups joined to this sugar- phosphate backbone at regular intervals. The sugar component of DNA does not have a 2'-hydroxyl group and is not as easily hydrolyzed in alkaline conditions, making the DNA backbone inher- ently more stable than that of RNA.

Rosalind Franklin (Watson and Crick)

Rosalind Franklin and Maurice Wilkins were using the powerful method of x-ray diffraction to analyze DNA fibers. Using the x-ray diffraction data obtained by Franklin and Wilkins, Watson and Crick proposed that DNA is composed of two polynucleotide strands entwined in the form of a right-handed double helix

Deamination (C->U)

Several nucleotide bases undergo deamination, a spontaneous loss of their exocyclic amino groups. The slow cytosine deamination reaction seems in- nocuous enough, but it is almost certainly the reason that DNA contains thymine rather than uracil. If DNA normally con- tained uracil, the recognition of U residues resulting from cytosine deamination would be more difficult, and unrepaired uracils would lead to permanent se- quence changes as they were paired with adenines

Copying Mechanism (Watson and Crick)

Suggested a copying mechanism with parent strands and daughter strands

B-DNA (Watson and Crick)

The Watson-Crick structure is known as B-form DNA, or B-DNA. B-DNA, the most common form in cells, has a wide major groove and a narrow minor groove. Has the C-2' endo pucker form. 3.4 Å apart, with 10.5 base pairs per turn.

Sugar-Phosphate backbone

The backbone of alternating sugars and phosphate groups is highly negatively charged. The hydro- philic backbones of alternating sugar and phosphate groups are on the outside of the helix, facing the sur- rounding water. Numer- ous bonds in the sugar-phosphate backbone can rotate, and thermal fluctuation can lead to bending, stretch- ing, and unpairing of the two strands.

4 different bases of deoxyribonucleotides and ribonucleotides

The bases are purines, with nine-membered rings, or pyrimidines, with six-membered rings, with numbering systems as shown. In DNA and RNA, the purines are adenine and guanine; in DNA, the pyrimidines are cytosine and thymine; in RNA, the pyrimidines are cytosine and uracil. he purine and pyrimidine bases common in DNA and RNA are conjugated ring systems, with alternating single and double bonds be- tween ring atoms. Resonance among atoms in the rings gives most of the bonds a partial double-bond character.

Purines (Adenine and Guanine)

The bases are purines, with nine-membered rings. The two purines are the same as those in DNA and RNA. Free pyrimidine and purine bases can exist in two or more forms, called tautomers, depending on pH. From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine residues: A+G=T+C. The purine and pyrimidine bases of both strands are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and relatively perpendicular to the long axis.

Hydrogen Bonds between bases (base pairing)

The second important mode of base interaction in nucleic acids is base pairing, which results from the hydrogen-bonding capacity of the ring nitrogens, ring carbonyl groups, and exocyclic. Hydrogen bonds between bases, involving the amino and carbonyl groups, permit a complementary association of two (and occasionally three or four) nucleic acid strands. G forms 3 bonds with C T forms 2 bonds with A

Chemical properties of purines and pyrimadines

The chemical properties of the purines and pyrimi- dines also give rise to two important modes of interaction between bases in nucleic acids. hydrophobic stacking, arises because the bases are hydrophobic and thus relatively insoluble in water at the near-neutral pH of the cell. Base stacking helps mini- mize the contact of the bases with water, and base- stacking interactions are very important in stabilizing the three-dimensional structure of nucleic acids. base pairing, which results from the hydrogen-bonding capacity of the ring nitrogens, ring carbonyl groups, and exocyclic (i.e., outside the ring structure) amino groups of the pyrimidines and pu- rines. Hydrogen bonds between bases, involving the amino and carbonyl groups, permit a complementary association of two (and occasionally three or four) nucleic acid strands. "A" hydrogen-bonds specifically with T (or U), and G with C (Figure 6-11 on page 184). These two types of base pairs predominate in double- stranded DNA and RNA (and thus are considered the canonical base pairs). Hydrophobic, van der Waals, and electrostatic interactions favor the alignment of bases in an aqueous solution or within a polynucleotide chain The purine and pyrimidine bases of both strands are stacked inside the double helix, with their hydrophobic and nearly planar ring structures very close together and relatively perpendicular to the long axis.

Nuceloside

The same molecule without the phosphate group is called a nucleoside. In nucleosides, the covalent joining of a base (at N-9 of purines and N-1 of pyrimidines) to the 1' carbon (C-1') of the pentose forms a glycosidic bond (specifi- cally, an N-􏰅-glycosyl bond), which involves the loss of a molecule of water. Nucleoside triphosphates are the activated pre- cursors of DNA and RNA synthesis. Furthermore, hydrolysis of nucleoside 5'-triphosphates, primarily ATP, provides the chemical energy to drive a wide variety of cellular reactions (see Chapter 3).

Phosphate

The same molecule without the phosphate group is called a nucleoside. To form a nucleotide, a phosphate group is covalently joined to the 5' carbon (C-5') of the pentose to form an ester, also with the concomitant loss of a water molecule. As we'll see, these 5'-to-3' links give every DNA or RNA chain a direction- ality, or polarity. The phosphate groups, with a pKa near 2, are completely ionized and negatively charged at pH 7, and the negative charges are generally neutralized by ionic interactions with positive charges on proteins, metal ions, or short, linear organic mole- cules

Uracil

The second major pyrimidine in RNA is uracil (U) instead of thymine. If DNA normally con- tained uracil, the recognition of U residues resulting from cytosine deamination would be more difficult, and unrepaired uracils would lead to permanent se- quence changes as they were paired with adenines

phosphodiester bond

The successive nucleotides of DNA and RNA are cova- lently joined through phosphate group "connectors" in which the 5'-phosphate group of one nucleotide unit is linked to the 3'-hydroxyl group of the next nucleotide, creating a phosphodiester bond. The backbone of alternating sugars and phosphate groups is highly negatively charged.

Major groove (double helix)

The two unequal surfaces formed by the twist of the helix are called the major groove and the minor groove. The major and minor grooves, where most interactions with proteins or other nucleic acids occur, are shown. One helical turn is about 10BPs