Chapter 5: DNA Replication, Repair, and Recombination
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Transcript of Chapter 5: DNA Replication, Repair, and Recombination
Chapter 5: DNA Chapter 5: DNA Replication, Repair, and Replication, Repair, and
Recombination Recombination
Maintenance of DNA SequencesMaintenance of DNA Sequences
Long Term Survival of Species Vs Survival of the Long Term Survival of Species Vs Survival of the IndividualIndividual
Maintenance of DNA SequencesMaintenance of DNA Sequences
Methods for Estimating Mutation RatesMethods for Estimating Mutation Rates► Rapid generation of bacteria makes possible to Rapid generation of bacteria makes possible to
detect bact w/ specific gene mutation detect bact w/ specific gene mutation Mutation in gene required for lactose Mutation in gene required for lactose
metabolism detected using indicator dyesmetabolism detected using indicator dyes► Indirect estimates of mutation rate: comparisons Indirect estimates of mutation rate: comparisons
of aa sequence of same protein across species of aa sequence of same protein across species ► Better estimates:Better estimates:
1.1. comparisions aa sequences in protein comparisions aa sequences in protein whose whose aa sequence does not matteraa sequence does not matter2.2. comparisions DNA sequences in regions comparisions DNA sequences in regions
of of genome that does not carry genome that does not carry critical infocritical info
Maintenance of DNA SequencesMaintenance of DNA Sequences
Many Mutations Are Deleterious & Many Mutations Are Deleterious & EliminatedEliminated
► Ea protein exhibits own characteristic rate of Ea protein exhibits own characteristic rate of evol which reflects probability that aa chg will evol which reflects probability that aa chg will be harmfulbe harmful
► 6-7 chgs harmful to cytochrome C6-7 chgs harmful to cytochrome C► Every aa chg harmful to histonesEvery aa chg harmful to histones
Maintenance of DNA SequencesMaintenance of DNA Sequences
Mutation Rates are Extremely LowMutation Rates are Extremely Low► Mutation rate in bact and mammals = 1 nucleotide chg/10Mutation rate in bact and mammals = 1 nucleotide chg/1099 nucleotides ea time nucleotides ea time
DNA replicatedDNA replicated► Low mutation rates essential for lifeLow mutation rates essential for life
Many mutations deleterious, cannot afford to accumulate in germ cellsMany mutations deleterious, cannot afford to accumulate in germ cells Mutation frequency limits number of essential proteins organism can encode ~60,000Mutation frequency limits number of essential proteins organism can encode ~60,000 Germ cell stability vs Somatic Cell StabilityGerm cell stability vs Somatic Cell Stability
Maintenance of DNA SequencesMaintenance of DNA Sequences
Multicellular Organisms Dependent upon Hi Fidelity Maintenance Afforded Multicellular Organisms Dependent upon Hi Fidelity Maintenance Afforded By:By:
1.1. Accuracy of DNA replication and distributionAccuracy of DNA replication and distribution2.2. Efficiency of DNA repair enzymesEfficiency of DNA repair enzymes
Maintenance of DNA SequencesMaintenance of DNA Sequences
High Fidelity DNA ReplicationHigh Fidelity DNA Replication► Error rate= 1 mistake/10Error rate= 1 mistake/1099 nucleotides nucleotides► Afforded by complementary base pairing and proof-reading capability of DNA polymeraseAfforded by complementary base pairing and proof-reading capability of DNA polymerase
Maintenance of DNA SequencesMaintenance of DNA Sequences
DNA Polymerase as Self Correcting EnzymeDNA Polymerase as Self Correcting Enzyme► Correct nucleotide greater affinity than incorrect nucleotideCorrect nucleotide greater affinity than incorrect nucleotide► Conformation Chg after base pairing causes incorrect nucleotide to dissociateConformation Chg after base pairing causes incorrect nucleotide to dissociate► Exonucleolytic proofreading of DNA polymeraseExonucleolytic proofreading of DNA polymerase
DNA molecules w/ mismatched 3’ OH end are not effective templates; DNA molecules w/ mismatched 3’ OH end are not effective templates; polymerase cannot extend when 3’ OH is not base pairedpolymerase cannot extend when 3’ OH is not base paired
DNA polymerase has separate catalytic site that removes unpaired DNA polymerase has separate catalytic site that removes unpaired residues at terminusresidues at terminus
Mechanism of DNA ReplicationMechanism of DNA Replication
General Features of DNA ReplicationGeneral Features of DNA Replication► SemiconservativeSemiconservative► Complementary Base PairingComplementary Base Pairing► DNA Replication Fork is AssymetricalDNA Replication Fork is Assymetrical► Replication occurs in 5’ 3’ DirectionReplication occurs in 5’ 3’ Direction
DNA ReplicationDNA Replication
Okazaki FragmentsOkazaki Fragments► DNA Primase uses rNTPs to synthesize short primers on lagging DNA Primase uses rNTPs to synthesize short primers on lagging
StrandStrand► Primers ~10 nucleotides long and spaced ~100-200 bp Primers ~10 nucleotides long and spaced ~100-200 bp ► DNA repair system removes RNA primer; replaces it w/DNADNA repair system removes RNA primer; replaces it w/DNA► DNA ligase joins fragmentsDNA ligase joins fragments
DNA ReplicationDNA Replication
DNA HelicaseDNA Helicase► Hydrolyze ATP when bound to ssDNA Hydrolyze ATP when bound to ssDNA
and opens up helix as it moves along and opens up helix as it moves along DNADNA
► Moves 1000 bp/secMoves 1000 bp/sec► 2 helicases: one on leading and one on 2 helicases: one on leading and one on
lagging strandlagging strand► SSB proteins aid helicase by SSB proteins aid helicase by
destabilizing unwound ss conformationdestabilizing unwound ss conformation
DNA ReplicationDNA Replication
SSB proteins help DNA helicase destabilizing ssDNA
DNA ReplicationDNA Replication
DNA Polymerase held to DNA by clamp regulatory proteinDNA Polymerase held to DNA by clamp regulatory protein► Clamp protein releases DNA poly when runs into dsDNAClamp protein releases DNA poly when runs into dsDNA► Forms ring around DNA helixForms ring around DNA helix► Assembly of clamp around DNA requires ATP hydrolysisAssembly of clamp around DNA requires ATP hydrolysis► Remains on leading strand for long time; only on lagging strand for short time when it Remains on leading strand for long time; only on lagging strand for short time when it
reaches 5’ end of proceeding Okazaki fragmentsreaches 5’ end of proceeding Okazaki fragments
DNA ReplicationDNA Replication
Replication Machine (1 x 10Replication Machine (1 x 106 6 daltonsdaltons)► DNA replication accomplished by DNA replication accomplished by
multienzyme complex that moves rapidly multienzyme complex that moves rapidly along DNA by nucleoside hydrolysisalong DNA by nucleoside hydrolysis
► Subunits include:Subunits include:(2) DNA Polymerases(2) DNA PolymeraseshelicasehelicaseSSBSSBClamp ProteinClamp Protein
► Increases efficiency of replicationIncreases efficiency of replication
DNA ReplicationDNA ReplicationOkazaki FragmentsOkazaki Fragments► RNA that primed synthesis of 5’ RNA that primed synthesis of 5’
end removedend removed► Gap filled by DNA repair Gap filled by DNA repair
enzymesenzymes► Ligase links fragments togetherLigase links fragments together
DNA ReplicationDNA Replication
Strand Directed Mismatch Repair SystemStrand Directed Mismatch Repair System► Removes replication errors not recognized by replication machineRemoves replication errors not recognized by replication machine► Detects distortion in DNA helixDetects distortion in DNA helix► Distinguishes newly replicated strand from parental strand by Distinguishes newly replicated strand from parental strand by
methylation of A residues in GATC in bactmethylation of A residues in GATC in bact► Methylation occurs shortly after replication occursMethylation occurs shortly after replication occurs► Reduces error rate 100XReduces error rate 100X► 3 Step Process3 Step Process
recognition of mismatchrecognition of mismatchexcision of segment of DNA containing mismatchexcision of segment of DNA containing mismatchresynthesis of excised fragmentresynthesis of excised fragment
DNA ReplicationDNA Replication
Strand Directed Mismatch RepairStrand Directed Mismatch Repair
DNA ReplicationDNA Replication
Strand Directed Mismatch Repair in HumansStrand Directed Mismatch Repair in Humans► Newly synthesized strand is preferentially nicked and can be Newly synthesized strand is preferentially nicked and can be
distinguish in this manner from parental stranddistinguish in this manner from parental strand► Defective copy of mismatch repair gene predisposed to cancerDefective copy of mismatch repair gene predisposed to cancer
DNA ReplicationDNA Replication
DNA TopoisomerasesDNA Topoisomerases► Reversible nuclease that covalently adds itself to DNA phosphate Reversible nuclease that covalently adds itself to DNA phosphate
backbone to break phosphodiester bondbackbone to break phosphodiester bond► Phosphodiester bond reforms as protein leavesPhosphodiester bond reforms as protein leaves► Two Types Two Types
Topoisomerase I- produces single stranded breakTopoisomerase I- produces single stranded break
Topoisomerase II- produces transient double stranded breakTopoisomerase II- produces transient double stranded break
DNA ReplicationDNA Replication
Topoisomerase ITopoisomerase I
DNA ReplicationDNA Replication
Topoisomerase IITopoisomerase II
DNA ReplicationDNA Replication
Eucaryotes vs ProcaryotesEucaryotes vs Procaryotes► Enzymology, fundamental features, replication fork geometry, and Enzymology, fundamental features, replication fork geometry, and
use of multiprotein machinery conserveduse of multiprotein machinery conserved► More protein components in Euk replication machineryMore protein components in Euk replication machinery► Replication must proceed through nucleosomes Replication must proceed through nucleosomes ► O. fragments in Euk ~200 bp as opposed to 1000-2000 ProO. fragments in Euk ~200 bp as opposed to 1000-2000 Pro► Replication fork moves 10X faster in ProReplication fork moves 10X faster in Pro
DNA ReplicationDNA Replication Initiation and Completion of DNA Replication in ChromosomesInitiation 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 ReplicationDNA Replication Initiation and Completion of DNA Replication in ChromosomesInitiation 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 ReplicationDNA Replication Initiation and Completion of DNA Replication in ChromosomesInitiation 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 ReplicationDNA Replication Initiation and Completion of DNA Replication in ChromosomesInitiation 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 ReplicationDNA Replication Initiation and Completion of DNA Replication in ChromosomesInitiation 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 ReplicationDNA Replication Initiation and Completion of DNA Replication in ChromosomesInitiation 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 ReplicationDNA Replication Initiation and Completion of DNA Replication in Initiation and Completion of DNA Replication in
ChromosomesChromosomes
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 ReplicationDNA Replication Initiation and Completion of DNA Replication in ChromosomesInitiation 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 RepairDNA 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 RepairDNA RepairTypes of DNA Damage: Base Loss and Base Types of DNA Damage: Base Loss and Base
ModificationModification
DepurinationChemical Modification Photodamage thymine
dimer
Chemical Modification by O2 free radicals
Deamination
DNA RepairDNA Repair
DNA GlycosylasesDNA GlycosylasesCleave glycosyl bond that connects base to backbone sugar to remove Cleave glycosyl bond that connects base to backbone sugar to remove
basebase>> 6 Different types including those that remove: 6 Different types including those that remove:
deaminated C’sdeaminated C’s different types of alkylated or oxidize basesdifferent types of alkylated or oxidize basesdeaminated A’sdeaminated A’s bases w/ open ringsbases w/ open ringsbases w/ C=Cbases w/ C=C
DNA RepairDNA Repair
Base Excision Repair
a. DNA glycosylase recognizes damaged base
b. Removes base leaving deoxyribose sugar
c. AP endonuclease cuts phosphodiester bkbone
d. DNA polymerase replaces missing nucleotide
e. DNA ligase seals nick
DNA RepairDNA Repair
Nucleotide Excision RepairNucleotide Excision Repaira.a. Bulky LesionBulky Lesion
b.b. RecognitionRecognition
c.c. Demarcation and unwindingDemarcation and unwinding
d.d. Assembly of Repair enzymesAssembly of Repair enzymes
e.e. Dual IncisionDual Incision
f.f. Release of Damaged Release of Damaged NucleotideNucleotide
g.g. Gap Filling DNA SynthesisGap Filling DNA Synthesis
DNA RepairDNA Repair
Chemistry of DNA Bases Facilitates Damage DetectionChemistry of DNA Bases Facilitates Damage DetectionRNA thot to be original genetic material A, C, G, URNA thot to be original genetic material A, C, G, U
Why U replaced w/ T?Why U replaced w/ T?
Deaminated C converted to UDeaminated C converted to U
DNA repair system unable to distinguish daminated C from U in RNADNA repair system unable to distinguish daminated C from U in RNA
DNA RepairDNA Repair
Repairing Double Stranded Breaks in DNARepairing 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 RepairDNA Repair
DNA Damage Can Activate Expression of Whole Sets of DNA Damage Can Activate Expression of Whole Sets of GenesGenes
► Heat Shock ResponseHeat Shock Response► SOS ResponseSOS Response
DNA RepairDNA Repair
DNA Damage Delays Progression of Cell CycleDNA Damage Delays Progression of Cell Cycle
DNA damage generates signals that block cell cycle progressionDNA damage generates signals that block cell cycle progression
Blocks occur to extend the time for DNA RepairBlocks occur to extend the time for DNA Repair
ATM ataxia telangiectasia- defects in gene encoding ATM proteinATM ataxia telangiectasia- defects in gene encoding ATM protein
RecombinationRecombination
► DNA sequences occasionally DNA sequences occasionally rearrangedrearranged
► Rearrangments may alter gene Rearrangments may alter gene structure as well as timing and level of structure as well as timing and level of expressionexpression
► Promote variationPromote variation
RecombinationRecombination
Two ClassesTwo Classes
1.1. General or Homologous RecombinationGeneral or Homologous Recombination
2.2. Site-Specific RecombinationSite-Specific Recombination
RecombinationRecombination
General or Homologous RecombinationGeneral or Homologous Recombination► Exchange btwn homologous DNA Exchange btwn homologous DNA
sequencessequences► Essential repair mechanismEssential repair mechanism► Essential for chromosomal segregationEssential for chromosomal segregation► Very PreciseVery Precise► Crossing over creates new combinations Crossing over creates new combinations
of DNA seq on ea chromoof DNA seq on ea chromo
RecombinationRecombination
Major Steps in General or Homologous RecombinationMajor Steps in General or Homologous Recombination
1.1. SynapsisSynapsis
2.2. Branch Chain MigrationBranch Chain Migration
3.3. Isomerization of Holliday JunctionIsomerization of Holliday Junction
4.4. Resolution Resolution
RecombinationRecombination
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
RecombinationRecombination
DNA Synapsis catalyzed by RecA ProteinDNA Synapsis catalyzed by RecA Protein► DNA strand from one helix has been exposed and its nucleotides DNA strand from one helix has been exposed and its nucleotides
made available for pairing w/ another molec= synapsismade available for pairing w/ another molec= synapsis► Initiated by endonuclease cutting two strands of DNA and 5’ end Initiated by endonuclease cutting two strands of DNA and 5’ end
chewed back to form ss 3’ endchewed back to form ss 3’ end► SSB proteins hold strands apartSSB proteins hold strands apart► RecA allows ssDNA to pair w/ homologous region of DNA=synapsisRecA allows ssDNA to pair w/ homologous region of DNA=synapsis
RecombinationRecombination
RecA Proteins also Facilitate Branch Chain MigrationRecA Proteins also Facilitate Branch Chain Migration► Unpaired region of one of the ss displaces paired region Unpaired region of one of the ss displaces paired region
of other ss moving the pointof other ss moving the point► RecA catalyzes unidirectional branch migration producing RecA catalyzes unidirectional branch migration producing
region of heteroduplex DNA 1000’s bp in lengthregion of heteroduplex DNA 1000’s bp in length
RecombinationRecombination
Holliday JunctionHolliday Junction► Two homolgous DNA helices paired and held together by reciprocal exchg of Two homolgous DNA helices paired and held together by reciprocal exchg of
two of the four strandstwo of the four strands► Two pairs of strands: one pair of crossing strands and one pair or noncrossingTwo pairs of strands: one pair of crossing strands and one pair or noncrossing► Isomerization leads to open structure where both pairs occupy equivalent Isomerization leads to open structure where both pairs occupy equivalent
positionspositions► Holliday junction resolved by cutting of helicesHolliday junction resolved by cutting of helices
RecombinationRecombination
Resolution of Holliday JunctionResolution of Holliday Junction
RecombinationRecombination
Site-Specific RecombinationSite-Specific Recombination► Mobile genetic elements move btwn nonhomologous sequencesMobile genetic elements move btwn nonhomologous sequences► Molibe genetic elementsMolibe genetic elements
size range 100s-1000s bpsize range 100s-1000s bp
found in nearly all cellsfound in nearly all cells
some represent viral sequencessome represent viral sequences
relics constitute significant portion of genome (repeat relics constitute significant portion of genome (repeat sequences)sequences)
RecombinationRecombination
Movement of Mobile Genetic ElementsMovement of Mobile Genetic Elements► Site specific recombo mediated by enzymes recognize short Site specific recombo mediated by enzymes recognize short
specific nucleotide sequences present in one or both of specific nucleotide sequences present in one or both of recombo DNA molecrecombo DNA molec
► No sequence homology requiredNo sequence homology required► Mobile genetic elements generally encode enzyme that Mobile genetic elements generally encode enzyme that
guides movement and special sites upon which enzyme actsguides movement and special sites upon which enzyme acts► Elements move by transposition or conservative mechanismsElements move by transposition or conservative mechanisms
RecombinationRecombination
Transpositional vs Conservative Site Specific Transpositional vs Conservative Site Specific RecombinationRecombination
► Transpositional= breakage rxns at ends of mobile DNA Transpositional= breakage rxns at ends of mobile DNA segments and attachment of those ends at one of many diff segments and attachment of those ends at one of many diff nonhomologous target sitesnonhomologous target sites
► Conservative= production of short heteroduplex joint and thus Conservative= production of short heteroduplex joint and thus requires short DNA sequence that is the same on both donor and requires short DNA sequence that is the same on both donor and recipient DNArecipient DNA
RecombinationRecombination
Transpositional Site Specific RecombinationTranspositional Site Specific Recombination► Can insert mobile genetic elements into any DNA sequenceCan insert mobile genetic elements into any DNA sequence► transposase acts on specific DNA seq at ea end of transposon transposase acts on specific DNA seq at ea end of transposon
disconnecting it from flanking DNA and inserting into new locationdisconnecting it from flanking DNA and inserting into new location► Transposons move only rarely (once every 10Transposons move only rarely (once every 1055 generations in bact) generations in bact)► 3 Types of Transposons3 Types of Transposons
RecombinationRecombination
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
RecombinationRecombination
Retroviral-like RetrotransposonsRetroviral-like Retrotransposons► Resemble retroviruses but lack protein Resemble retroviruses but lack protein
coatcoat► Transcription of transposon into RNATranscription of transposon into RNA► Transcript translated by host encodes RT Transcript translated by host encodes RT
that produces ds DNA that produces ds DNA ► Linear ds DNA integrates into site on Linear ds DNA integrates into site on
chromo using integrase also encoded by chromo using integrase also encoded by transposontransposon
RecombinationRecombination
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
RecombinationRecombination
Different Transponable Elements Predominate in Different Different Transponable Elements Predominate in Different OrganismsOrganisms
► Bacterial transposons are of DNA only type w/ a few nonretroviral Bacterial transposons are of DNA only type w/ a few nonretroviral transposonstransposons
► Yeast main mobile elements are retroviral retrotransposonsYeast main mobile elements are retroviral retrotransposons► Drosophilia and humans contain all three types of tranposable Drosophilia and humans contain all three types of tranposable
elementselements