Chapter28Notes 11.45.23 PM

Chapter28Notes 11.45.23 PM - Page 1 of 30 Chapter 28 Notes...

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Unformatted text preview: Page 1 of 30 Chapter 28 Notes Biochemistry 461 Fall 2010 CHAPTER 28: DNA Structure, Replication, Repair, and Recombination LECTURE TOPICS 1) DNA STRUCTURE 2) DNA-PROTEIN INTERACTIONS 3) DNA TOPOISOMERASES 4) DNA REPLICATION: The process 5) DNA RECOMBINATION 6) DNA MUTATIONS 7) DNA REPAIR KEY CONCEPTS: ! DNA structure is dynamic. It can be bent, kinked, unwound, and have different helical structures. ! Proteins interact with DNA in all biological activities involving DNA. ! DNA must be unwound to replicate. ! Topoisomerases catalyze changes in supercoiled state of DNA. ! DNA replication has three distinct phases (initiation, elongation, and termination). Termination is different at telomeres of eucaryotic chromosomes ! DNA replication is very accurate (1x10-8 mistakes/base). ! DNA molecules can “recombine” if they have similar sequences. ! Mutations have several causes and involve base sequence changes. ! DNA repair corrects errors using highly evolved correction systems. Page 2 of 30 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 B-DNA 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: Watson-Crick Model Real X-ray structure of B-DNA 36° turn per base pair 28-42° turn per base pair paired bases in same plane propeller twisting (bases not parallel) adjacent base pairs parallel base roll structural regularities not dependent on base sequence structure details are sequence specific (Provides 3-D uniqueness for proteinDNA interactions) ! B-DNA structure is dynamic. The helix can be bent into an arc or supercoiled with little effect on structure (BIG bend figure). [This permits circle formation, wrapping of DNA around proteins, and packing in a cell]. DNA can be kinked (exs: AAAA's in a row or protein binding). ! Major and minor grooves occur in B-DNA because glycosidic bonds of a base pair are asymmetrically oriented with respect to the helix axis. (Fig.27.7) ! Different functional groups are located in these grooves. These groups are either 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. Page 3 of 30 DNA-B Structure: Fig.27.8 DNA-B BIG BEND Fig.31-2 Page 4 of 30 DNA bases: not in same plane - Propeller twist (Fig.27.9) Major and minor grooves (Fig.27.7) H-donors in blue H-acceptors in red Page 5 of 30 DNA can exist in several structural forms. Three (A, B, and Z) are discussed. Page 6 of 30 MAJOR FEATURES OF A and Z-DNA STRUCTURES (See Figs and Table 27.1) A-DNA STRUCTURE: A-DNA is "-helical double helix (right-handed). Wider and shorter (per turn of helix) than B-DNA. Base pairs tilted 19° (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. [Figs. 31-7, 31-9]. The 2'-OH of ribose in RNA won't fit in a "B-DNA" type structure (steric hindrance).[Fig. 27-6] Page 7 of 30 ! RNA-RNA double strands (like in RNA hairpin loops) and RNA-DNA hybrids are A-form. A and Z DNA’s compared to B-DNA: (Figs.27.4 and 27.10) Z-DNA STRUCTURE: Another form of DNA found in DNA with alternating GC base pairs (GCGCGC). [Fig. 31-9]. 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. Page 8 of 30 DNA-PROTEIN INTERACTIONS NON-SPECIFIC INTERACTIONS: Deoxyribonuclease I binds electrostatically to DNA at minor groove. Bonds (over a whole turn of the helix) are salt bridges between PO4 backbone and arginine and lysine NH2 groups in DNase I. Since DNase I interacts with DNA PO4 groups, little or no discrimination between cut sites is possible (i.e., cuts are non-specific with regard to sequence). [See BIG BEND DNA-B on p.3] 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. For example, ! Type II restriction endonucleases (Examples, EcoRV and EcoRI) cut double-stranded DNA with high specificity, cleaving recognition sites with two-fold symmetry at identical sites on each strand. These restriction enzymes are dimers of identical subunits which bind DNA with coincident two-fold symmetry axes of the protein and the DNA recognition site. [NOTE: See Text Chapter 9, pp. 245-252 for EcoRV-protein interactions] Features of sequence specific DNA-protein interactions: ! Specific interaction is with functional groups on bases of DNA recognition sites. ! DNA is usually kinked when proteins are bound, and helix is unwound, 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 backbone of DNA. Page 9 of 30 Eco RV restriction enzyme interactions with its DNA recognition site: ! Eco RV recognition site has two-fold symmetry (Fig.9.37) ! Eco RV asymmetric dimer binds on one side of the DNA helix. [Fig 31-10, 4th Ed.] Page 10 of 30 ! Eco RV opens the DNA helix [Fig 31-10, 4th Ed.]and kinks the DNA about 50° (Fig.9.40) ! Specific H-bonds form between functional groups on bases on each DNA strand and amino acid side chains of Eco RV $-turns. (Fig.9.39) ! Eco RV has four structural elements that are evolutionarily-conserved , being also found in other Type II restriction enzymes. (Fig.9.44) Page 11 of 30 Eco RI restriction enzyme interactions with its DNA recognition site: ! ! Dipole chargecharge interactions ! H-bonds with amino acids 6 H-bonds with amino acids Page 12 of 30 DNA TOPOLOGY: DNA-BINDING PROTEINS ALTER THE TOPOLOGY OF DNA ! Negative supercoiled circular DNA is compact and is energetically favored. Most DNA in cells has negative supercoiled (right-handed) “superhelices”. Superhelices are underwound. This facilitated DNA helix unwinding for replication, recombination, transcription, etc. ! Positive supercoils (left-handed) make opening the helix more difficult. ! The topology of DNA (state of supercoiling) can be changed by unwinding or winding supercoils. Changes in linking number result in different DNA topoisomers. Changes require cutting one or both DNA strands. [Figs.27.2, 31-16, 31-17, 31-18] Different states of DNA supercoiling (negative and positive) Page 13 of 30 ! Topisomerase enzymes can DNA convert + to - supercoils Topoisomerase II [2 strands cut] [right-handed supercoils] (DNA Gyrase - uses ATP) Topoisomerase I [1 strand cut] [left-handed supercoils] (NOTE: Helicase in DNA replication adds positive supercoils, makes NO cuts, and uses ATP) DNA 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 ATP energy required for this reaction. A 5'-P at cleavage site is activated by covalent linking to a tyrosine on the enzyme. Then a 3'-OH nucleophilic attack ligates the cut strand of DNA after removing one or more supercoils. (Fig. 27.22) [formally, this is adding positive supercoils] Page 14 of 30 Topoisomerase I cuts one strand (cuts, rotates, ligates) ! (Fig.27.22) Topoisomerase II (DNA Gyrase in DNA replication): A class of proteins that add negative supercoils to DNA using ATP hydrolysis for energy (9 kcal/mole) (Fig.27.24). In replication, 200bp of DNA wrap around the gyrase holoenzyme molecule, 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. Two DNA gyrase inhibitors are nalidixic acid (prevents strand cutting and rejoining) and novobiocin (blocks ATP binding) are. Page 15 of 30 ! DNA ligase joins free 3'-OH ends with a 5'-P group of adjacent bases (can think of as a "nicked" DNA strand) forming a new phosphodiester bond. New 3'-5' phospodiester bond ! NAD+ or ATP react with DNA ligase to give an enzyme-linked AMP that is then transferred to the 5'-P end of the DNA. This AMP-activated 5'-P is subjected to a nucleophilic attack by the free 3'-OH end. AMP is the leaving group and a new 3'-5' phosphodiester bond results. )Two high energy P-bonds are used in the complete reaction with either NAD+ or ATP). [Fig. 31-12, and p.761-2 of 5th Ed.] Page 16 of 30 DNA REPLICATION: The polymerases [** Review Ch.5 Figures, pp. 13-15] DNA POLYMERASES: (E.coli examples Pol I and Pol III) ! DNA POLYMERASE I (Pol I): HAS THREE DIFFERENT ACTIVITIES 1) Template-Directed Polymerase activity (5' to 3') (Fig.5.22) Synthesizes DNA from 5' to 3'. E. coli Pol I enzyme is simplest and best understood DNA polymerase. Though not responsible for most chromosomal DNA replication, it has functions in DNA replication and DNA repair. A 103kd monomeric, a ZN++ enzyme. A Processive enzyme, i.e., don't need association-dissociation step for each nucleotide added. About 20 bases added before enzyme falls off DNA and another one takes its place. [Error rate = 1 x 10-4] 2) Proofreading 3' to 5' Exonuclease Activity [Fig. 31-24] ! Hydrolyses nucleotides in 3' to 5' direction at 3'-OH end of DNA primer if wrong nucleotide. ! Editing (proofreading) activity helps prevent errors during DNA replication. ! If a mutation causes reduced proofreading, get higher mutation rates; if increased, can reduce mutation rates). [Misses wrong base at rate = 1 x 10-4] Page 17 of 30 3) Error-Correcting 5' to 3' Exonuclease Activity [Fig. 31-25] ! Hydrolyses, DNA from 5' to 3' direction, if a free 5'-P end of a double-stranded DNA is encountered. ! Cuts are at 5'-terminal nucleotide or several nucleotides from 5'-end. Corrects errors in preexisting DNA. ! Involved in repair of DNA and in removal of RNA primer used for DNA replication. Place concept of DNA polymerase accuracy (error rates) in context of Flow of genetic information [Review Ch.5, Part 2 Figures, p.21- Central Dogma] Pol I Active Sites and Structure: DNA polymerase I has a different active sites for each activity. [4th Ed., Figs. 31-26, 31-27] and (5th Ed. Figs.27.11-15) 5' to 3' Polymerase domain Page 18 of 30 ! Role of two Mg++ in Pol I 5' to 3' polymerase is to coordinate 3'OH with dNTP and with 2-Asp of Pol I. (Fig.27.12) ! Correct base-pairing of incoming dNTP is anchored by H-bonds between minor groove H-acceptors (same place for A-T and G-T) and Arg and Gln of Pol I. (Fig.27.13) ! Correct dNTP binding induces conformational shift that gives tight binding pocket for dNTP and template DNA. (Fig.27.14) Page 19 of 30 ! 3' to 5' Exonuclease activity of Pol I when added base is released from polymerase site and “edited” at distant site. (Fig.27.15) ! DNA POLYMERASES II AND III. E. coli Mutants lacking Pol I grow and replicate DNA normally. DNA polymerase II (function unknown) and III found 15 years after Pol I. Both have (per cell) lower number of molecules and activity than Pol I. Both have 5' to 3' DNA polymerase and proofreading 3' to 5' exonuclease activity. [but no 5' - 3' exonuclease function] ! DNA POLYMERASE III catalyzes most of E. coli chromosomal DNA replication. It is a multi-subunit, asymmetric dimer enzyme. It adds deoxyribonucleotides at a rate of 1000 bases/sec to the primer. This is about 100 times faster is more processive than Pol I (thousands of bases, compared to about 20). The " subunits are catalytic while a $2 dimer forms a sliding clamp with a hole in the middle which can accomodate the DNA template-primer complex. (Figs.27.30, 31) Page 20 of 30 DNA Polymerase III $2 dimer i DNA Polymerase III - DNA Complex STUDY HINT: COMPARE PROPERITES OF DNA POLYMERASES I, II, AND III DNA REPLICATION: The process INITIATION OF DNA REPLICATION: ! Replicating DNA molecules appear to be in 2 (theta) structures showing E. coli DNA as circular during replication. DNA unwinding and replication takes place at 2 replication forks and proceeds in both direction simultaneously. ! Replication starts at a unique site (OriC, 245 bp) from two replication forks. (Fig.27.25) Binding of dnaA protein (and others)initiates replication. Page 21 of 30 THE DNA REPLICATION CYCLE: Replication is complex. Many proteins in addition to polymerases are involved. One replicating DNA strand (leading strand) is synthesized continuously and the other (lagging strand) is synthesized discontinuously. The processive Pol III dimer connects about 1000 bases.(Figs.27.3 and 27) Apparent directions of DNA synthesis Actual (5' to 3') direction of DNA synthesis LEADING STRAND SYNTHESIS (elongation) ! Helicase unwinds DNA. (ATP hydrolysis required - introduces positive supercoils.) ! SSB protein binds to the parental single strands as they are unwound. ! DNA gyrase introduces negative supercoils to relieve torsional strain (ATP hydrolysis required). Page 22 of 30 ! 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. (Fig.27.26) ! Pol III dimer adds deoxyribonucleotides to the RNA primer. TERMINATION OF DNA REPLICATION : Pol I cleaves off RNA primers and fills in gaps (both leading and lagging strands); DNA ligase seals gaps. [Fig. 31-38] Page 23 of 30 LEADING AND LAGGING STRAND SYNCHRONOUS SYNTHESIS (Figs. 27.32, 33) LAGGING STRAND SYNTHESIS [(elongation) The lagging strand appears to form a loop to reverse polarity, presenting itself to one subunit of the Pol III dimer in the correct polarity for DNA synthesis. After connecting about 1000 bases, a new primer is needed for another 1000 base fragment, etc. Page 24 of 30 Termination of Eucaryotic DNA replication: The Problem - it’s a linear chromosome, so how to complete the ends?? (Can’t just ligate ends and get a circle as with E. coli chromosome; see Fig 37-15, 4th Ed.) ! Eucaryotic “Telomere ” structure (Fig.27.35) DNA replication leaves one incomplete end [Fig 37-15] Telomere synthesis by telomerase (Fig.27.36) Page 25 of 30 DNA RECOMBINATION Recombination mechanism, Cre recombinase, and Holliday Junctions (Fig.6.31 and Figs 27.37, 39) Page 26 of 30 MUTATIONS are DNA base changes that arise from mismatched bases in DNA caused by: ! replication errors, chemical mutagens, ultraviolet light (sunlight!) SPECIFIC MUTAGENS ! ! ! ! Base analogs (cause mispairing and transitions). chemicals that modify bases (cause transitions). intercalating agents (cause insertions or deletions). ultraviolet light (causes pyrimidine dimers, then get replication errors). ARE MUTAGENIC AGENTS also CARCINOGENS? ! Ames test (Salmonella His- to His+) Rare C:A base pair Chemical mutagens Nitrous acid: deaminates adenine (get A-T to G-C transitions) Page 27 of 30 Base analog (5-BU) Intercalating agent (causes insertions - frameshifts) Aflatoxin: activated by cytochrome P450 and adds to N-7 of guanine. (Causes G-C to T-A transversions) Thymine dimers- adjacent bases in same DNA strand Page 28 of 30 DNA REPAIR: replication errors are 1 in108, but fixed by repair to 1 in 1010 bases) Repair pathways (Fig.27.47) ! T-T dimer repair 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) Page 29 of 30 ! uracil removal (uracil comes 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. 31-44] ! mismatch repair 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, stomach, and uterine cancers. [Fig. 31-45] Page 30 of 30 Triplet repeats occur in eucaryotic chromosomes in tandem arrays. Addition of more than normal number of them cause genetic diseases. [Exs.: Huntington disease, other neurological diseases known too] (Fig.27.52) ...
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