chapter28 - Chapter 28 DNA Replication, Repair, and...

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Unformatted text preview: Chapter 28 DNA Replication, Repair, and Recombination DNA replication, damage, and repair Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. DNA is flexible Watson-Crick DNA structure (B form DNA) was derived from DNA fibers which gives average structure of the DNA. X-ray analysis of crystals of DNA give much more detailed structural information and showed that two other forms of DNA also exist. Sequences of alternating purine and pyrimidine bases (synthesized) form Z type DNA helix. Z = zig-zagging of PO4 along surface of helix Z-DNA is left-handed (counterclockwise) helix Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. ost DNA is in B form (Watson-Crick) M - plane of purine-pyrimidine base pair is perpendicular to axis of helix nder conditions of low humidity, DNA will assume A form double helix U - plane of purine-pyrimidine base pair is not perpendicular to axis of helix (19 tilt). A form helix results from "sugar puckering" of deoxyribose ring to a C3' endo position. Because of 2' OH group, double stranded RNA does not fit into B form (WatsonCrick) helix. Double stranded regions of RNA is A form. Propeller twist enhances the stacking of bases (Base pairs are often not perfectly coplanar) Major and minor grooves in DNA: important for proteins to interact (Dickerson et al., 1982) >90% DNA-Modifying Enzymes A. Sequence-independent enzymes DNase I Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Degrades DNA nonspecifically to short `oligonucleotides' DNase I is an endonuclease (cuts DNA from both ends) Cuts single and double stranded DNA Positively charged lysine and arginine residues of DNase I bind to phosphate groups of DNA Catalyzes the hydrolysis of DNA phosphodiester bonds DNA Ligase Catalyzes the formation of a phosphodiester bond between the 3' OH group at one end of a DNA strand and the 5' phosphate group at the end of another DNA strand. (no substrate specificity) Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Covalently joins two molecules of DNA: reverse of DNase I In bacteria NAD+ is the source of energy for phosphodiester bond formation, while in mammals ATP is the energy donor Ends to be joined must be double-stranded. Single stranded DNA is not a substrate for the enzyme Enzyme mechanism involves: 1. formation of covalent enzyme-AMP complex 2. transfer of AMP to 5' end of DNA to form DNA-AMP complex 3. nucleophilic attack of 3' OH of second DNA strand on the activated phosphate of DNA-AMP complex DNA Topology ~ Topoisomerase I and II (DNA Gyrase) Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Supercoiling of DNA - one mechanism of compacting DNA 0.34 nm/bp x 3,000,000,000 base pairs (human) = 1 meter / average nucleus is 5 !m in diameter - consider linear 260 bp DNA duplex B-form DNA gives 25 helical turns (260 bp/10.4 bp per helical turn) If ends of this DNA are joined "relaxed circle" (DNA ligase) If this DNA is unwound by two helical turns before joining DNA ends, the circle could assume two structures unwound circle or negative superhelix. Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. ligate writhe Supercoiled DNA Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Topologically different supercoiled forms can be described by three parameters Lk = linking number = number of times one strand of DNA winds around the other in the right hand direction Tw = twisting number = number of helical turns in DNA Wr = writhing number = number of turns of superhelix; right hand = negative Lk = Tw + Wr (Tw is usually very close to # bp/10.4 in the cell) Changes in linking number usually caused by change in Wr (supercoiling) but not much change in Tw Supercoiled DNA molecules are more compacted than relaxed circular DNA molecules Topoisomers Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. (DNA molecules with same number of DNA, but differing in linking number) Lk = linking number = number of times one strand of DNA winds around the other in the right hand direction Tw = twisting number = number of helical turns Wr = number of turns of superhelix Lk = Tw + Wr topoisomers Topoisomers can be interconverted only by cutting and rejoining. Topoisomerases interconvert topoisomers of DNA - Two types of topoisomerases Type I relaxes DNA by increasing the Lk number by one with each catalytic cycle -Thermodynamically favorable reaction Mechanism 1. Cleave one strand of DNA: 3' end of broken DNA strand is anchored to tyrosine residue 2. Passage of DNA strand through break 3. Reseal DNA break Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. DNA gyrase (topoisomerase II) Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. - introduces negative supercoils into circular DNA by reducing Lk number (by two) - energy consuming reaction driven by ATP hydrolysis - 5' phosphate groups are anchored to tyrosine residues (like topoisomerase I) - DNA gyrase is essential to Replication and Transcription Demonstrated by two antibiotics Cyproflaxacin and Nalidixic Acid interferes with breaking and joining of DNA strands Novobiocin blocks ATP binding to gyrase B. Sequence specific enzymes: Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Restriction Endonucleases: mechanism used by bacteria to prevent infection by bacteriophage - a primitive a immune system ethylation protects host bacterial DNA, restriction endonuclease cleaves phage DNA m EXAMPLE: EcoRV restriction endonuclease (E. coli) - recognizes hexanucleotide palindromic sequence - enzyme binds as a dimer, opens major groove for sequence recognition, cleaves between T and A by hydrolysis of a phosphodiester bond. DNA replication Semi-conservative required Reaction 5' direction 3' complementary Steps of DNA replication Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Klenow fragment DNA polymerase I lacking 5' end first DNA polymerase identified DNA polymerase I Core polymerase 5'-3'exonuclease 3'-5'exonuclease Klenow Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. DNA polymerases Two metal ions (typically Mg2+) participate in the polymerase reaction (interact with primer and dNTP) Specificity of replication is dictated by complementarity of shape between bases E. coli chromosomal replication Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Replication structures can be seen by electron microscopy Theta structures Replication forks Replication starts at a unique site (~245 bp) on the E. coli chromosome Ori C Two replication forks progress around the chromosome in opposite directions The two replication forks meet at a unique location (tre) and replication ends DNA replication = RNA polymerase (DnaG) (RNA pol does not need primer) OH : 5~6 nucleotides III NA removed (exonuclease)/DNA filled by DNA polymerase I R igated by DNA ligase L Leading strand and lagging strand (Okazaki fragments) Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. DNA polymerases catalyze synthesis of DNA strand in 5' to 3' direction but replication forks appear to be synthesizing one strand in 5' to 3' direction and the other strand in 3' to 5' direction. Problem was solved by Okazaki who showed that one strand is synthesized continuously (leading strand) and the other strand is synthesized discontinuously (lagging strand). Discontinuous synthesis leads to production of DNA fragments of 500-1000bp known as Okazaki fragments. DNA Polymerase III synthesizes both the leading strand and lagging strand. It is a highly processive enzyme and incorporates many thousands of nucleotides before letting go of DNA. Lagging strand synthesis involves synthesis of short RNA primers by a specific RNA polymerase called primase. DNA polymerase then elongates these RNA primers to generate Okazaki fragments. Gaps between Okazaki fragments are filled and RNA is removed by DNA pol I. Finally, the DNA fragments on the lagging strand are joined by DNA ligase. Molecules involved in E. coli DNA polymerization elicase (DnaB): Hexamer to H open DNA by ATP hydrolysis ingle-strand-binding protein S (SSB): keep the strands separated rimase (DnaG) P NA polymerase III D Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. DNA polymerase III holoenzyme Two core enzymes : polymerase 2: a ring structure that serves as a sliding clamp : proofreading exonuclease (3' to 5') Character: 1. Very active (high catalytic potency) - 1000~2000 nucleotides per second (100~200 turns pass through / sec) 2. Processivity - catalyze many consecutive reactions without releasing substrates (role of the 2 dimer) 3. High fidelity Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. 2 dimer DNA replication is highly coordinated Replication of the leading and lagging strands is coordinated by the looping out of the lagging strand to form a structure that act like a trombone slide. When the polymerase reaches a region that has been replicated, the sliding clamp is released and a new loop is formed. RNA primer is removed (5' to 3' exonuclease activity) and the gap is filled by DNA polymerase I. Fragments are connected by DNA ligase. Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Prokaryotic vs Eukaryotic DNA synthesis Eukaryotic DNA synthesis Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Multiple origins (~30,000; not sharply defined) 6 billion bp, 23 pairs of chromosomes in human <--> 4.6 million bp, 1 chromosome in bacteria Assembly of "origin of replication complexes" (ORC: homologous to DnaA) ~ licensing factors including the helicase (Mcm2-7) ~ SSB (replication protein A) ~ polymerases Polymerase switching: "Replication factor C" (RFC) displaces polymerase and attracts "proliferating cell nuclear antigen" (PCNA: a sliding clamp homologous to the 2 subunit). PCNA binds to polymerase (more processive) to continue replication. DNA replication is linked to the cell cycle (cell division): Mitosis (M) takes place only after DNA synthesis (S). Checkpoints control the progression along the cycle: "cyclin" and "cyclin-dependent protein kinases" (cdk) regulate replication through interlocking mechanisms. - role in cancer - DNA synthesis Eukaryotic DNA synthesis Eukaryotes chromosomes are linear The lagging strand would have an incomplete 5' end after the removal of the RNA primer (replication would shorten the chromosome) RNA primer Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. The ends of chromosomes (telomeres) contain hundreds of tandem repeats of a six-nucleotide sequence (AGGGTT) Telomerase, a polymerase (reverse transcriptase) carrying its own RNA template replicates telomeres - roles in cancer and aging Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Errors in DNA replication Expansion of repeats of 3 nucleotides is found in Huntington's disease CAG repeats in "huntingtin": 6~3136~82 repeats poly-glutamine (prone to aggregates). Looping out of the repeats from the template may increase the number of copies in replication. Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Mutations in DNA Three main types of mutation: 1. Substitution (point mutation) either transition (common) or transversion (less common) Transitions: AG (purinepurine) or CT (pyrpyr) Transversions: AC, AT, GC, or GT (purinepyr) missense (different amino acid) nonsense (stop) silent (no change), etc. 2. Deletion 3. Insertion frame shift, etc. Chemical agents / light can modify nucleotide bases in DNA Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. 1. Reactive oxygen species such as hydroxyl radical reacts (oxidation) with guanine to form 8-oxoguanine. Base pairs with adenine rather than cytosine causing transversion. 2. Deamination of adenine to hypoxanthine Hypoxanthine pairs with cytosine rather than thymine causing transition 3. Aflatoxin (fungal toxin) 4. Ultraviolet light covalently link adjacent pyrimidine bases thymine dimer Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. <attack amino group (Alkylation) to cause GC to TA transversion> (crosslinked) DNA repair roofreading P ex. subunit of E. coli DNA pol III 3' to 5' exonulease Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Direct reversal 1. Thymine dimers: The photoreactivation process directly reverses the damage (cleaves the dimer) by the action of the photoreactivating enzyme called DNA photolyase 2. Methylation: ex. methylation of guanine bases is directly reversed by the protein methyl guanine methyl transferase (MGMT) Single strand damage 1. Base-excision repair: repairs damage to a single nucleotide 2. Nucleotide-excision repair 3. Mismatch repair ouble strand damage D 1. Non-homologous end joining (NHEJ): Ku70/80 heterodimers help seal the break 2. Recombinational repair (homologous recombination repair) Single strand damage repair ase-excision repair: B ex. AlkA flips damaged DNA to remove 3-methyladenine (base) with its glycosylase activity Nucleotide-excision repair: Nucleotides Mismatch repair systems: In E. coli, MutS detects the mismatch and MutS-MutL recruits endonuclease (MutH) containing thymine dimmers are removed by uvrABC excinuclease AP endonuclease Deoxyribose phosphodiesterase DNA pol I ~ DNA ligase (from Augusto-Pinto et al. 2003) Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Thymine instead of Uracil in DNA for enhanced fidelity: Repair of deaminated cytosine (= uracil) to cytocine (base-excision repair) U in DNA is repaired to C Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Many cancers are caused by defective repair of DNA Genes for DNA repair proteins are often tumor-suppressor genes -UvrABC in xeroderma pigmentosum -hMSH2 (MutS) and hMLH1 (MutL) in hereditary nonpolyposis colorectal cancer (HBPCC, Lynch syndrome) -p53 (sense damage, promote repair/apoptosis) in various cancers including Li-Fraumeni syndrome Cancers often treated by damaging DNA - cancer cells divide frequently - cancer cells often have defects in DNA-repair pathways Screening for mutagens: Ames Test Carcinogenicity can be tested using the Ames test Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Uses specific Salmonella bacteria that have a mutation (substitute, frameshift) in one of the genes for the biosynthesis of histidine (also lacks repair system), which cannot grow on histidine-minus media. Mutagenicity of compound is determined by number of "revertant" bacteria whose gene is mutated back to be able to synthesize histidine to grow on histidine-minus media. Potential mutagens can be "activated" by incubation with liver extract (recall aflatoxin activation by cytochrome P450) DNA recombination Roles in replication, repair, and antibody diversity -RecA: strand invasion -Cre recombinase Homologous recombination repair DNA double strand break Homologous duplex DNA Strand invasion Repair synthesis Holliday junctions Resolution (reactome.org) Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. Eukaryotic Chromosomal DNA Structure (chapter 31, p903) istones DNA binding protein found in chromatin H (chromatin = entire complex of a cell's DNA and associated protein) istones are basic proteins with relative mass of 11~20 kd H Arg+, Lys+ -rich; note that DNA is negatively charged Five types of histones H1, H2A, H2B, H3, H4 Histones are highly evolutionarily conserved hey are important in the formation of nucleosomes T Chromatin structure (EM) Nucleosome Edited by Foxit Reader Copyright(C) by Foxit Software Company,2005-2007 For Evaluation Only. 2 copies each H2A, H2B, H3, H4 & ~200 bp of DNA Nucleosome core particle: histone octamer + 145 bp DNA Nucleosome + H1 Nucleosome core (Histone talis: may be modified to control transcription) H1 binds to the outside of the core particle and the linker DNA. Nucleosome compacts DNA by a factor of 6~7 (~680 --> ~110 ). "Higher order" structures compact DNA by a factor of 104. Nucleosomes must be disassembled and reassembled for replication and transcription. ...
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