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Chapter 5: DNA Chapter 5: DNA Replication, Repair, Replication, Repair, and Recombination and Recombination

Chapter 5: DNA Replication, Repair, and Recombination

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Page 1: Chapter 5: DNA Replication, Repair, and Recombination

Chapter 5: DNA Chapter 5: DNA Replication, Repair, and Replication, Repair, and

Recombination Recombination

Page 2: Chapter 5: DNA Replication, Repair, and 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

Page 3: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 4: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 5: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 6: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 7: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 8: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 9: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 10: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 11: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 12: Chapter 5: DNA Replication, Repair, and Recombination

DNA ReplicationDNA Replication

SSB proteins help DNA helicase destabilizing ssDNA

Page 13: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 14: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 15: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 16: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 17: Chapter 5: DNA Replication, Repair, and Recombination

DNA ReplicationDNA Replication

Strand Directed Mismatch RepairStrand Directed Mismatch Repair

Page 18: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 19: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 20: Chapter 5: DNA Replication, Repair, and Recombination

DNA ReplicationDNA Replication

Topoisomerase ITopoisomerase I

Page 21: Chapter 5: DNA Replication, Repair, and Recombination

DNA ReplicationDNA Replication

Topoisomerase IITopoisomerase II

Page 22: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 23: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 24: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 25: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 26: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 27: Chapter 5: DNA Replication, Repair, and Recombination

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)

Page 28: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 29: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 30: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 31: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 32: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 33: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 34: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 35: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 36: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 37: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 38: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 39: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 40: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 41: Chapter 5: DNA Replication, Repair, and Recombination

RecombinationRecombination

Two ClassesTwo Classes

1.1. General or Homologous RecombinationGeneral or Homologous Recombination

2.2. Site-Specific RecombinationSite-Specific Recombination

Page 42: Chapter 5: DNA Replication, Repair, and 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

Page 43: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 44: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 45: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 46: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 47: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 48: Chapter 5: DNA Replication, Repair, and Recombination

RecombinationRecombination

Resolution of Holliday JunctionResolution of Holliday Junction

Page 49: Chapter 5: DNA Replication, Repair, and Recombination

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)

Page 50: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 51: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 52: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 53: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 54: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 55: Chapter 5: DNA Replication, Repair, and Recombination

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

Page 56: Chapter 5: DNA Replication, Repair, and Recombination

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