CHAPTER 28: DNA STRUCTURE, REPLICATION AND REPAIR
Biochemistry 461

Notes - pdf

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    A)   DNA STRUCTURE:  (Models vs X-ray Structures; Static vs Dynamic Models)
                    * B-DNA
                    * A-DNA (also in RNA-RNA double strands and RNA-DNA hybrids)
                    * Z-DNA.
    B)   DNA-PROTEIN INTERACTIONS (sequence specific vs non-specific)

• DNA TOPOISOMERASES  (bring about changes in state by cutting and sealing DNA strands)

• DNA structure is dynamic - it can be bent, kinked, unwound, have different helical structures, as shown primarily by x-ray diffraction studies.
• Proteins interact with DNA during all the biological functions with which DNA is associated.
• Deoxyribonuclease I and EcoRI and RV restriction enzyme interactions with DNA show how these proteins recognize and bind to DNA.
• Topoisomerases catalyze changes in the supercoiled state of DNA molecules.
• DNA replication has three distinct phases (initiation, elongation, and termination).
• Not only is DNA replication accurate (1x10^-8 mistakes/base) but mistakes that do occur (as a result of "mutagens", etc.) are usually repaired by highly evolved error correction mechanisms.
 

A) DNA STRUCTURE: (Models vs X-ray Structures; Static vs Dynamic Structures)

• B-DNA: MODEL vs X-RAY STRUCTURE. Watson-Crick -helix B-DNA structure (very regular) came from model building based on x-ray diffraction data from DNA fibers consisting of parallel oriented DNA molecules.
• REAL STRUCTURE: X-ray structure of crystals of 12-bp DNA (dodecamer) looks mostly like Watson-Crick B-form helix, but there are some irregularities compared to W-C model: [27-9]

• DNA can be kinked (AAAA's in a row or protein binding can kink).
• Major and minor grooves of B-DNA occur because the glycosidic bonds of a bp are asymmetrically oriented with respect to the helix axis.
• Functional groups located in major and minor grooves. All these groups can be hydrogen bond donors (d) or hydrogen bond acceptors (a).
• The major groove is more accessible for high specificity interactions with proteins and chemical reagents, being physically much larger and having more functional groups than the minor groove.
• A-DNA is -helical double helix (right-handed), but wider and shorter (per turn of helix) and base pairs are tilted 19^o (not perpendicular) from axis of helix. Most differences between A and B helix are due to different puckering of sugar (C'-₃-endo in A-DNA, C'₂-endo in B-DNA. 2'-OH of ribose in RNA won't fit in a "B-DNA" type structure (steric hindrance). RNA-RNA double strands (like in RNA hairpin loops) and RNA-DNA hybrids are A-form.
• Z-DNA. Another form of DNA deduced from x-ray structural analysis of double-stranded oligomer of alternating GC base pairs (GCGCGC). [Fig. 27-10] It is a left-handed helix with a zig-zag phosphate backbone and only one deep groove. Z-DNA is favored also by C₅-methylation of cytosine which occurs in eucaryotes (where evidence of Z-DNA comes from binding of antibodies to Z-DNA). The actual biological functions of Z-DNA are not known.
• COMPARISONS OF MAJOR FEATURES OF A, B AND Z-DNA STRUCTURES: [Table 27-1]

• NON-SPECIFIC INTERACTIONS: Deoxyribonuclease I binds electrostatically to DNA in minor groove via salt bridges between PO₄ backbone and arginine and lysine NH₂ groups in DNase I over a whole turn of the helix. Since the protein-DNA interaction is with PO₄, there is little or no discrimination of cut sites relative to specific DNA sequences (i.e., cuts are non-specific with regard to sequence).
• SEQUENCE-SPECIFIC INTERACTIONS: In general, the major groove is more accessible for high specificity interactions with proteins and chemical reagents, being physically much larger and having more functional groups than the minor groove. EcoRIand EcoRV restriction endonucleases cut double-stranded DNA with high specificity, cleaving recognition sites with two-fold symmetry at identical sites on each strand. The enzymes are dimers of identical subunits which bind DNA with coincident two-fold symmetry axes of the protein and the DNA recognition site.

   

As a rule these are the types of interactions which confer sequence specificity to DNA-protein interactions:

• sequence specificity requires interaction with the bases of DNA recognition sites.

• DNA is kinked when the enzymes are bound and the helix is unwound slightly allowing protein -helices into major groove for specific interactions.

• Specific H-bonds between functional groups on bases on each DNA strand and amino acid side chains of enzyme -helices or loops from -turns are formed. "Dipolar" helices of EcoRI also interact electrostatically with the P0₄ backboneof DNA.

• DNA ligase joins free 3'-OH ends with a 5'-P group of adjacent bases in a "nicked" strand of a "D.S.-DNA". A new phosphodiester bond is formed. NAD⁺ or ATP react with DNA ligase to give an enzyme-bound AMP which is transferred to the 5'-P of the DNA. The AMP "activates" the 5'-P which is subjected to a nucleophilic attack by the 3'-OH. AMP is the leaving group and a new 3'5 phosphodiester bond results. Two high energy P-bonds are used in the complete reaction.
• Negative Supercoiling of circular DNAs is energetically favored and leads to more compact DNA. Most naturally occurring DNA has Negative supercoils (right-handed) which are underwound, giving "superhelices" which facilitate unwinding of the double-helix for replication, recombination and transcription. Positive supercoils (left-handed) make opening the helix more difficult. The topology of DNA can be changed by unwinding or winding into positive or negative supercoils. This changes the linking number resulting in different topoisomers of a DNA molecule (this requires cutting one or both DNA strands.

• There are two types of Topoisomerases. Topoisomerase I catalyzes relaxation of negative supercoils by (1) cleavage of one DNA strand; (2) passage of a segment of DNA through the break, and (3) resealing the break. No high energy phosphate donor is required for this thermodynamically-favorable reaction.A 5'-P at the cleavage site is activated by covalent linking to a tyrosine on the enzyme, prior to a 3'-OH nucleophilic attack which restores the intact D.S.-DNA after removing one or more negative supercoils. The enzyme has a hole in the middle which can accomodate a DNA molecule. [Topo II Fig. 27-24]

• DNA Gyrase adds negative supercoils to DNA using ATP hydrolysis for energy (9 kcal/mole). 200bp of DNA wrap around the gyrase holoenzymemolecule, ATP is bound, and each strand is cut (staggered cuts) and covalently linked to different tyrosines to "anchor" the DNA. Then a segment of D.S.-DNA passes through the cut, the cut ends are religated, and ATP hydrolysis releases the DNA from the gyrase. Two negative supercoils are added with each catalytic step as a result of D.S.-DNA passage through a break in both strands. Nalidixic acid (prevents strand cutting and rejoining) and novobiocin (blocks ATP binding) are DNA gyrase inhibitors.

• Topoisomerase Summary

• DNA POLYMERASE I (Pol I): AN ENZYME WITH THREE DIFFERENT ACTIVITIES [Fig. 31-26]

• Synthesizes DNA from 5'to 3' making 3'-5'-phosphodiester bonds.
• Discovered in 1955 - (E. coli enzyme). Simplest and best understood of the DNA polymerases, not responsible for most chromosomal DNA replication in E. coli, but functions in vivo both in DNA replication and DNA repair.
• One 103kd polypeptide monomer, a ZN⁺⁺ enzyme.
• Requires 4-dNTPs, Mg⁺⁺ and a template-primer DNA complex.
• Primer must have free 3'-OH end. PPi (product) hydrolysis drives reaction.
• 20 nucleotide units are added by enzyme before it falls off DNA and a new one is needed (A Processive enzyme, i.e., don't need an association-dissociation step for each nucleotide added).

• Hydrolyses nucleotides from the 3'5' direction at 3'-OH end of DNA primer chain if the 3'-nucleotide is improperly base-paired.
• This is an editing or proofreading activity which prevents errors during DNA replication.
• If proofreading reduced, get higher mutation rates, (if activity increased have lower mutation rates).

• Hydrolyses, DNA from 5' to 3' direction usually ahead of new DNA synthesis on primer if the DNA is double-stranded. Cuts are made at 5'-terminal nucleotide or several nucleotides from 5'-end.
• Corrects errors in preexisting DNA. Is involved in repair of DNA and in removal of RNA primer used for DNA replication.
• Polymerase accuracy (error rates)

• DNA polymerase I has three separate activities and three different active sites. Protease cleavage gives a 36-kd fragment with 5'3' exonuclease activity and a 67-kd fragment with both 5'3' polymerase and 3'5 editing activities. The 67-kd fragment has two domains and a deep crevice into which double-stranded DNA fits well. [Figs. 31-26, Pol I Klenow Fragment]

• E. coli Mutants lacking Pol I have normal growth and replication.

• DNA polymerase II and III were discovered 15 years after Polymerase I. Both have lower amounts and activity (per cell) than Pol I.
• Pol II and Pol III catalyze 5' to 3' DNA polymerase reaction (template-directed, as for Pol I) using a primer with free 3'-OH.
• Both Pol II and Pol III have proofreading 3' to 5' exonuclease activity.
• Biological role of Pol II unknown, but Pol III is responsible for chromosomal DNA replication (at least 20 other proteins - not polymerases - are also involved in E. coli DNA replication).

• Replicating DNA molecules appear to be in theta structures which show E. coli DNA to be circular during replication with DNA unwinding and replication taking place at replication forks (*).
• Replication starts at a unique site (OriC) on the E. coli chromosome and proceeds in both direction simultaneously from two replication forks. OriC is 245 bp long and has 3 tandemly oriented, nearly identical sequences and 4 binding sites for DNA A protein, whose binding initiates replication.
• One replicating DNA strand (leading strand) is synthesizedcontinuously and the other (lagging strand) is synthesized discontinuously. Discovery of Okazaki fragments gave proof.

• RNA primase (a specific RNA polymerase) synthesizes a primer of about 5 bases long. The RNA primer is later removed (and the gap filled in) by Pol I.

• DNA Polymerase III [Pol III] (a multi-subunit, asymmetric dimer enzyme) which adds deoxyribonucleotides (1000 bases/sec) to the RNA primer. Pol III is about 10 times faster and more processive (thousands of bases, compared to about 20) than Pol I. The subunits are catalytic while a ₂dimer forms a sliding clamp with a hole in the middle which can accomodate the DNA template-primer complex.

DNA POLYMERASE I, II, AND III REVIEW

THE DNA REPLICATION CYCLE

• The replication cycle is most complex. Many proteins are involved. The details are best learned by studying Figures [27-32], [Synthesis Initiation], [Termination] and [Table of Replication Proteins].

1) dna A,B and C proteins bind to OriC.

2) Helicase (dna B) unwinds DNA. (ATP hydrolysis required - introduces positive supercoils.)

3) SSB protein binds to the parental single strands as they are unwound.

4) DNA gyrase introduces negative supercoils to relieve torsional strain (ATP hydrolysis required).

5) RNA primase synthesizes the RNA primer.

6) Pol III (a dimer) adds deoxyribonucleotides to the RNA primer.

LAGGING STRAND SYNTHESIS [FIGURES 27-32 and 27-33]

• Maybe the lagging strand loops around to present to one subunit of the dimeric Pol III, a DNA strand which is in the proper polarity for DNA synthesis by a processive Pol III dimer for about 1000 bases, then a new primer is made for another 1000 base fragment, etc.

• Pol I cleaves off RNA primers and fills in gaps (both leading and lagging strands); DNA ligase seals gaps.

1) errors in replication (1x10^-10). [Figs. 27-41]

2) chemical mutagens [Fig. 27-43]

3) ultraviolet light [Fig. 27-46]

SPECIFIC MUTAGENS

a) Base analogs (which cause mispairing and transitions). [Fig. 24-42]

b) chemical agents which chemically modify bases (cause transitions). [Fig. 27-45]

c) intercalating agents (cause insertions or deletions). [Fig. 27-44]

d) ultraviolet light (causes pyrimidine dimers). [Fig. 27-46]

e) eucaryotic triplet repeat expansions [Fig. 27-52]

E) DNA REPAIR PROCESSES

• Excision of lesion (T-T dimers) new DNA synthesis by Pol I and ligase to seal nick.Xeroderma pigmentosum is caused by a defect in the exonuclease (or any of 8 other genes) which removes pyrimidine dimers. [Fig. 27-49]
• Removal of uracil (from deamination of cytosine) in DNA is by specific uracil N-glycosidase which leaves thymine alone (no cleavage). Thus the C₅-CH₃ group of thymine may allow discrimination of it from the deamination product (Uracil) of cytosine which must be removed. This is another DNA fidelity enhancer. [Fig. 27-50]

• Mismatch repair in E.coli is carried out by several proteins which are related to human proteins in which mutations are related to incidence of colorectal cancers. [Fig. 27-51]

F) IS A MUTAGEN ALSO A CARCINOGEN: The Ames test measures the mutagenic properties of a chemical in a bacterial system. The chemical is also incubated with a liver extract in whic cytochrome P450 activities can modifiy the chemical, making it more or less muatagenic. This treatment mimics what might occur in a mammalian system, and so can suggest that a mutagen could also be carcinogenic in humans. [Fig. 27-53]