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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
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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
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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
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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:
1.
2.
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
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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
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Mechanism of DNA Replication
General Features of DNA Replication
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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
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DNA Replication
SSB proteins help DNA helicase destabilizing ssDNA
DNA Replication
DNA Polymerase held to DNA by clamp regulatory protein
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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
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DNA Replication
Okazaki Fragments
RNA that primed synthesis of 5’ end
removed
► Gap filled by DNA repair enzymes
► Ligase links fragments together
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DNA Replication
Strand Directed Mismatch Repair System
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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
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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
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DNA Replication
Topoisomerase I
DNA Replication
Topoisomerase II
DNA Replication
Eucaryotes vs Procaryotes
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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
Deamination
Depurination
Photodamage thymine dimer
Chemical Modification by O2 free radicals
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
deaminated A’s
bases w/ C=C
different types of alkylated or oxidize bases
bases w/ open rings
DNA 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 Repair
Nucleotide Excision Repair
a.
b.
c.
d.
e.
f.
g.
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
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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
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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
1.
2.
3.
4.
Steps in General or Homologous Recombination
Synapsis
Branch Chain Migration
Isomerization of Holliday Junction
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
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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
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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
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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
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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
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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
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