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Chapter 5: DNA Replication, Repair, and Recombination

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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 ... – PowerPoint PPT presentation

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


1
Chapter 5 DNA Replication, Repair, and
Recombination
2
Maintenance of DNA Sequences
  • Long Term Survival of Species Vs Survival of the
    Individual

3
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

4
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

5
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

6
Maintenance of DNA Sequences
  • Multicellular Organisms Dependent upon Hi
    Fidelity Maintenance Afforded By
  • Accuracy of DNA replication and distribution
  • Efficiency of DNA repair enzymes

7
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

8
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

9
Mechanism of DNA Replication
  • General Features of DNA Replication
  • Semiconservative
  • Complementary Base Pairing
  • DNA Replication Fork is Assymetrical
  • Replication occurs in 5 3 Direction

10
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

11
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

12
DNA Replication
SSB proteins help DNA helicase destabilizing ssDNA
13
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

14
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

15
DNA Replication
  • Okazaki Fragments
  • RNA that primed synthesis of 5 end removed
  • Gap filled by DNA repair enzymes
  • Ligase links fragments together

16
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

17
DNA Replication
  • Strand Directed Mismatch Repair

18
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

19
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

20
DNA Replication
  • Topoisomerase I

21
DNA Replication
Topoisomerase II
22
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

23
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

24
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

25
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 oris
  • Individual oris spaced at intervals of
    30,000-300,000 bp

26
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 oris takes 1 hr
  • BrdU experiments
  • Highly condensed chromatin replicates late while
    less
  • condensed regions replicate early
  • Housekeeping and cell specific genes

27
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)

28
DNA Replication Initiation and Completion of DNA
Replication in Chromosomes
  • Mammalian DNA Sequences that Specify Initiation
    of Replication
  • 1000s 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

29
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

30
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

31
DNA Repair
  • Despite 1000s 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

32
DNA Repair Types of DNA Damage Base Loss and
Base Modification
Chemical Modification
Photodamage thymine dimer
Depurination
Chemical Modification by O2 free radicals
Deamination
33
DNA Repair
  • DNA Glycosylases
  • Cleave glycosyl bond that connects base to
    backbone sugar to remove base
  • gt 6 Different types including those that remove
  • deaminated Cs different types of alkylated or
    oxidize bases
  • deaminated As bases w/ open rings
  • bases w/ CC

34
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

35
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

36
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

37
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
38
DNA Repair
  • DNA Damage Can Activate Expression of Whole Sets
    of Genes
  • Heat Shock Response
  • SOS Response

39
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

40
Recombination
  • DNA sequences occasionally rearranged
  • Rearrangments may alter gene structure as well as
    timing and level of expression
  • Promote variation

41
Recombination
  • Two Classes
  • 1. General or Homologous Recombination
  • 2. Site-Specific Recombination

42
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

43
Recombination
  • Major Steps in General or Homologous
    Recombination
  • 1. Synapsis
  • 2. Branch Chain Migration
  • 3. Isomerization of Holliday Junction
  • 4. Resolution

44
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

45
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
    DNAsynapsis

46
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 1000s bp in
    length

47
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

48
Recombination
  • Resolution of Holliday Junction

49
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)

50
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

51
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

52
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

53
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

54
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

55
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

56
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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