Chapter 5: DNA Replication, Repair, and Recombination

Maintenance of DNA Sequences Long Term Survival of Species Vs Survival of the Individual

Maintenance of DNA Sequences Methods for Estimating Mutation Rates Rapid generation of bacteria makes possible to detect bact w/ specific gene mutation Mutation in gene required for lactose metabolism detected using indicator dyes Indirect estimates of mutation rate: comparisons of aa sequence of same protein across species Better estimates: 1. comparisions aa sequences in protein whose aa sequence does not matter 2. comparisions DNA sequences in regions of genome that does not carry critical info

Maintenance of DNA Sequences Many Mutations Are Deleterious & Eliminated Ea protein exhibits own characteristic rate of evol which reflects probability that aa chg will be harmful 6-7 chgs harmful to cytochrome C Every aa chg harmful to histones

Maintenance of DNA Sequences Mutation Rates are Extremely Low Mutation rate in bact and mammals = 1 nucleotide chg/109 nucleotides ea time DNA replicated Low mutation rates essential for life Many mutations deleterious, cannot afford to accumulate in germ cells Mutation frequency limits number of essential proteins organism can encode ~60,000 Germ cell stability vs Somatic Cell Stability

Maintenance of DNA Sequences Multicellular Organisms Dependent upon Hi Fidelity Maintenance Afforded By: Accuracy of DNA replication and distribution Efficiency of DNA repair enzymes

Maintenance of DNA Sequences High Fidelity DNA Replication Error rate= 1 mistake/109 nucleotides Afforded by complementary base pairing and proof-reading capability of DNA polymerase

Maintenance of DNA Sequences DNA Polymerase as Self Correcting Enzyme Correct nucleotide greater affinity than incorrect nucleotide Conformation Chg after base pairing causes incorrect nucleotide to dissociate Exonucleolytic proofreading of DNA polymerase DNA molecules w/ mismatched 3’ OH end are not effective templates; polymerase cannot extend when 3’ OH is not base paired DNA polymerase has separate catalytic site that removes unpaired residues at terminus

Mechanism of DNA Replication General Features of DNA Replication Semiconservative Complementary Base Pairing DNA Replication Fork is Assymetrical Replication occurs in 5’ 3’ Direction

DNA Replication Okazaki Fragments DNA Primase uses rNTPs to synthesize short primers on lagging Strand Primers ~10 nucleotides long and spaced ~100-200 bp DNA repair system removes RNA primer; replaces it w/DNA DNA ligase joins fragments

DNA Replication DNA Helicase Hydrolyze ATP when bound to ssDNA and opens up helix as it moves along DNA Moves 1000 bp/sec 2 helicases: one on leading and one on lagging strand SSB proteins aid helicase by destabilizing unwound ss conformation

DNA Replication SSB proteins help DNA helicase destabilizing ssDNA

DNA Replication DNA Polymerase held to DNA by clamp regulatory protein Clamp protein releases DNA poly when runs into dsDNA Forms ring around DNA helix Assembly of clamp around DNA requires ATP hydrolysis Remains on leading strand for long time; only on lagging strand for short time when it reaches 5’ end of proceeding Okazaki fragments

DNA Replication Replication Machine (1 x 106 daltons) DNA replication accomplished by multienzyme complex that moves rapidly along DNA by nucleoside hydrolysis Subunits include: (2) DNA Polymerases helicase SSB Clamp Protein Increases efficiency of replication

DNA Replication Okazaki Fragments RNA that primed synthesis of 5’ end removed Gap filled by DNA repair enzymes Ligase links fragments together

DNA Replication Strand Directed Mismatch Repair System Removes replication errors not recognized by replication machine Detects distortion in DNA helix Distinguishes newly replicated strand from parental strand by methylation of A residues in GATC in bact Methylation occurs shortly after replication occurs Reduces error rate 100X 3 Step Process recognition of mismatch excision of segment of DNA containing mismatch resynthesis of excised fragment

DNA Replication Strand Directed Mismatch Repair

DNA Replication Strand Directed Mismatch Repair in Humans Newly synthesized strand is preferentially nicked and can be distinguish in this manner from parental strand Defective copy of mismatch repair gene predisposed to cancer

DNA Replication DNA Topoisomerases Reversible nuclease that covalently adds itself to DNA phosphate backbone to break phosphodiester bond Phosphodiester bond reforms as protein leaves Two Types Topoisomerase I- produces single stranded break Topoisomerase II- produces transient double stranded break

DNA Replication Topoisomerase I

DNA Replication Topoisomerase II

DNA Replication Eucaryotes vs Procaryotes Enzymology, fundamental features, replication fork geometry, and use of multiprotein machinery conserved More protein components in Euk replication machinery Replication must proceed through nucleosomes O. fragments in Euk ~200 bp as opposed to 1000-2000 Pro Replication fork moves 10X faster in Pro

DNA Replication Initiation and Completion of DNA Replication in Chromosomes DNA Replication Begins at Origins of Replication Positions at which DNA helix first opened In simple cells ori defined DNA sequence 100-200 bp Sequence attracts initiator proteins Typically rich in AT base pairs

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Bacteria Single Ori Initiation or replication highly regulated Once initiated replication forks move at ~400-500 bp/sec Replicate 4.6 x 106 bp in ~40 minutes

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Eukaryotic Chromosomes Have Multiple Origins of Replication Relication forks travel at ~50 bp/sec Ea chromosome contains ~150 million base pairs Replication origins activate in clusters or replication units of 20-80 ori’s Individual ori’s spaced at intervals of 30,000-300,000 bp

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Eukarotic DNA replication During S phase Ea chromo replicates to produce 2 copies that remain joined at centromeres until M phase S phase lasts ~8 hours Diff regions on same chromosomes replicate at distinct times during S phase Replication btwn 2 ori’s takes ~ 1 hr BrdU experiments Highly condensed chromatin replicates late while less condensed regions replicate early Housekeeping and cell specific genes

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Replication Origins Well Defined Sequences in Yeast ARS autonomously replicating sequence ARS spaced 30,000 bp apart ARS deletions slow replication ORC origin recognition complex marks replication origin binds Mcm (DNA helicase) Cdc6 (helicase loading factor)

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Mammalian DNA Sequences that Specify Initiation of Replication 1000’s bp in length Can function when placed in regions where chromo not too condensed Human ORC required for replication initiation also bind Cdc6 and Mcm proteins Binding sites for ORC proteins less specific

DNA Replication Initiation and Completion of DNA Replication in Chromosomes New Nucleosomes Assembled Behind Replication Fork lg amt of new histone protein required during replication 20 repeated gene sets (H1, H2A, H2B, H3, H4) Histones syn in S phase ( transcription, degradation) Histone proteins remarkably stable Remodeling complexes destabilize DNA histone interface during replication CAFs (chromatin assembly factors) assist in addition of new nucleosome behind replication fork

DNA Replication Initiation and Completion of DNA Replication in Chromosomes Telomerase Replicates Ends of Chromosomes Telomere DNA sequences contain many tandem repeat sequences Human telomere sequence GGGTTTA extends 10,000 nucleotides Telomerase= special reverse transcriptase Telomerase elongates repeat sequence recognizing tip of G-rich strand uses RNA template that is a component of enzyme itself Protruding 3’ end loops back to hid terminus and protect it from degradative enzymes

DNA Repair Despite 1000’s of alterations that occur in DNA ea day, few are retained as mutations Efficient reapir mechanisms Impt of DNA repair highlighted by: # of genes devoted to DNA repair mutation rates as a function of inactivation or loss of DNA repair gene Defects in DNA repair associated w/ several disease states

DNA Repair Types of DNA Damage: Base Loss and Base Modification Chemical Modification Photodamage thymine dimer Depurination Chemical Modification by O2 free radicals Deamination

DNA Repair DNA Glycosylases Cleave glycosyl bond that connects base to backbone sugar to remove base > 6 Different types including those that remove: deaminated C’s different types of alkylated or oxidize bases deaminated A’s bases w/ open rings bases w/ C=C

DNA Repair Base Excision Repair DNA glycosylase recognizes damaged base Removes base leaving deoxyribose sugar AP endonuclease cuts phosphodiester bkbone DNA polymerase replaces missing nucleotide DNA ligase seals nick

DNA Repair Nucleotide Excision Repair Bulky Lesion Recognition Demarcation and unwinding Assembly of Repair enzymes Dual Incision Release of Damaged Nucleotide Gap Filling DNA Synthesis

DNA Repair Chemistry of DNA Bases Facilitates Damage Detection RNA thot to be original genetic material A, C, G, U Why U replaced w/ T? Deaminated C converted to U DNA repair system unable to distinguish daminated C from U in RNA

DNA Repair Repairing Double Stranded Breaks in DNA Nonhomologous end-joining repair original DNA sequence is altered during repair (deletions or insertions) Homologous end-joining repair general recombination mechanism; info transferred from intact strand

DNA Repair DNA Damage Can Activate Expression of Whole Sets of Genes Heat Shock Response SOS Response

DNA Repair DNA Damage Delays Progression of Cell Cycle DNA damage generates signals that block cell cycle progression Blocks occur to extend the time for DNA Repair ATM ataxia telangiectasia- defects in gene encoding ATM protein

Recombination DNA sequences occasionally rearranged Rearrangments may alter gene structure as well as timing and level of expression Promote variation

Recombination Two Classes 1. General or Homologous Recombination 2. Site-Specific Recombination

Recombination General or Homologous Recombination Exchange btwn homologous DNA sequences Essential repair mechanism Essential for chromosomal segregation Very Precise Crossing over creates new combinations of DNA seq on ea chromo

Recombination Major Steps in General or Homologous Recombination 1. Synapsis 2. Branch Chain Migration 3. Isomerization of Holliday Junction 4. Resolution

Recombination General or Homologous Recombination Guided by Base Pairing Interactions Cross over of DNA from different chromosomes ds helices break and two broken ends join opp. partners to reform intact ds helices Exchange occurs only if there is extensive sequence homology No nucleotides are altered at site of exchange; no loss or gain

Recombination DNA Synapsis catalyzed by RecA Protein DNA strand from one helix has been exposed and its nucleotides made available for pairing w/ another molec= synapsis Initiated by endonuclease cutting two strands of DNA and 5’ end chewed back to form ss 3’ end SSB proteins hold strands apart RecA allows ssDNA to pair w/ homologous region of DNA=synapsis

Recombination RecA Proteins also Facilitate Branch Chain Migration Unpaired region of one of the ss displaces paired region of other ss moving the point RecA catalyzes unidirectional branch migration producing region of heteroduplex DNA 1000’s bp in length

Recombination Holliday Junction Two homolgous DNA helices paired and held together by reciprocal exchg of two of the four strands Two pairs of strands: one pair of crossing strands and one pair or noncrossing Isomerization leads to open structure where both pairs occupy equivalent positions Holliday junction resolved by cutting of helices

Recombination Resolution of Holliday Junction

Recombination Site-Specific Recombination Mobile genetic elements move btwn nonhomologous sequences Molibe genetic elements size range 100s-1000s bp found in nearly all cells some represent viral sequences relics constitute significant portion of genome (repeat sequences)

Recombination Movement of Mobile Genetic Elements Site specific recombo mediated by enzymes recognize short specific nucleotide sequences present in one or both of recombo DNA molec No sequence homology required Mobile genetic elements generally encode enzyme that guides movement and special sites upon which enzyme acts Elements move by transposition or conservative mechanisms

Recombination Transpositional vs Conservative Site Specific Recombination Transpositional= breakage rxns at ends of mobile DNA segments and attachment of those ends at one of many diff nonhomologous target sites Conservative= production of short heteroduplex joint and thus requires short DNA sequence that is the same on both donor and recipient DNA

Recombination Transpositional Site Specific Recombination Can insert mobile genetic elements into any DNA sequence transposase acts on specific DNA seq at ea end of transposon disconnecting it from flanking DNA and inserting into new location Transposons move only rarely (once every 105 generations in bact) 3 Types of Transposons

Recombination DNA Only Transposons Move by DNA breakage and joining “cut and paste” mechanism Inverted repeat recognized at ends and brought together forming loop Insertion catalyzed by transposase occurs at random sites through staggered breaks Break resealed but breakage and repair often alters DNA sequence resulting in mutations at site of excision

Recombination Retroviral-like Retrotransposons Resemble retroviruses but lack protein coat Transcription of transposon into RNA Transcript translated by host encodes RT that produces ds DNA Linear ds DNA integrates into site on chromo using integrase also encoded by transposon

Recombination Nonretroviral Retrotransposons L1 or LINE for long interspersed nuclear element L1 RNA synthesis Endonuclease attached to L1 RT and L1 RNA Endonuclease nicks target DNA at insertion site Released 3’ OH end used as primer for RT that generates ssDNA copy of element linked to target Leads to synthesis of second DNA strand that is inserted where original nick was made

Recombination Different Transponable Elements Predominate in Different Organisms Bacterial transposons are of DNA only type w/ a few nonretroviral transposons Yeast main mobile elements are retroviral retrotransposons Drosophilia and humans contain all three types of tranposable elements