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CHAPTER 5 DNA REPLICATION, REPAIR AND RECOMBINATION

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Title: CHAPTER 5 DNA REPLICATION, REPAIR AND RECOMBINATION


1
CHAPTER 5 DNA REPLICATION, REPAIR AND
RECOMBINATION
  • THE MAINTENANCE OF DNA SEQUENCES
  • DNA REPLICATION MECHANISMS
  • THE INITIATION AND COMPLETION OF DNA REPLICATION
    IN CHROMOSOMES
  • DNA REPAIR
  • GENERAL RECOMBINATION
  • SITE-SPECIFIC RECOMBINATION

2
THE MAINTENANCE OF DNA SEQUENCES
  • Mutation Rates Are 1/109 bp
  • Inherent fidelity of DNA polymerase is 1/106 bp
  • Other mechanisms, proofreading and repair are
    necessary
  • Low Mutation Rates Are Necessary for Life as We
    Know It

3
Mutation rates are relatively constant but
proteins evolve at different rates
  • Proteins evolve different at different rates
    depending on structural and functional constrains
  • In some proteins most changes interfere with
    function
  • In others many sequence changes are tolerated

4
DNA REPLICATION MECHANISMS
  • Base-pairing Underlies DNA Replication and DNA
    Repair
  • DNA Replication is always in the 5-to-3
    Direction
  • The DNA Replication Fork Is Asymmetrical
  • Fidelity of DNA Replication Requires Several
    Proofreading Mechanisms

5
Base-pairing Underlies DNA Replication and DNA
Repair
6
DNA replication is semiconservative
7
Fidelity of DNA Replication Requires Proofreading
8
Many polymerases contain separate 3-gt5 editing
domains
9
The DNA Replication Fork Is Asymmetrical
  • Leading and lagging strands have different
    requirements

10
Accesssory proteins
  • DNA Primase Synthesizes Short RNA Primer
    Molecules on the Lagging Strand
  • Helicases - Open Up the DNA Double Helix in Front
    of the Replication Fork
  • Single strand binding proteins keep ssDNA out of
    trouble
  • Clamp subunits tether A Moving DNA Polymerase to
    the DNA
  • The Proteins at a Replication Fork Cooperate to
    Form a Replication Machine

11
DNA Primase Synthesizes Short RNA Primer
Molecules on the Lagging Strand
12
Helicases - Open Up the DNA Double Helix
13
Single strand binding proteins keep ssDNA out of
trouble
14
Clamp subunits tether A Moving DNA Polymerase to
the DNA
15
Clamp loading and unloading on the lagging strand
16
The replication machine - expanded
17
The replication machine - trombone model
18
The eucaryotic fork is like the procaryotic but
with more specialization
19
Other factors operate away from the replication
fork
  • A Strand-directed Mismatch Repair System Removes
    Replication Errors That Escape from the
    Replication Machine
  • DNA Topoisomerases Prevent DNA Tangling During
    Replication
  • All topoisomerases form transient covalent
    phospho-tyrosine bonds to DNA backbone
  • Type 1 topoisomerase nicks only one strand -
    unwinds only
  • Type 2 topoisomerase nicks both strands - unwinds
    and untangles - Makes DNA ethereal

20
A Strand-directed Mismatch Repair System Removes
Replication Errors That Escape from the
Replication Fork
21
DNA Topoisomerase Prevents DNA Tangling During
Replication
22
Type 1 topoisomerase nicks only one strand -
unwinds only
23
Type 2 topoisomerase nicks both strands -
Unwinds and Untangles - Makes DNA ethereal
24
THE INITIATION OF DNA REPLICATION
  • DNA Synthesis Begins at Replication Origins
  • BacteriaHave a Single Origin
  • Eucaryotic Chromosomes Contain Multiple Origins
  • In Eucaryotes DNA Replication Takes Place During
    Only One Part of the Cell Cycle
  • Different Regions on the Same Chromosome
    Replicate at Distinct Times in S Phase
  • Highly Condensed Chromatin Replicates Late,While
    Genes in Less Condensed Chromatin Tend to
    Replicate Early

25
DNA Synthesis Begins at Replication Origins
26
BacteriaHave a Single Origin
27
Initiation and mismatch repair in bacteria are
both controlled by methylation
28
Origins can be mapped by pulse labeling
29
Different DNA regions have distinct timings of
replication during the S phase of the eukaryotic
cell cycle
30
Well-defined DNA Sequences Serve as Replication
Origins in a Simple Eucaryote, the Budding Yeast
  • A Large Multisubunit Complex Binds to Eucaryotic
    Origins of Replication
  • The Mammalian DNA Sequences That Specify the
    Initiation of Replication Have Been Difficult to
    Identify
  • New Nucleosomes Are Assembled Behind the
    Replication Fork

31
Yeast Origins have been identified by genetic
means
32
A Large Multisubunit Complex Binds to Eucaryotic
Origins of Replication
33
Chromatin is assembled following replication forks
34
COMPLETION OF DNA REPLICATION IN CHROMOSOMES
  • Telomerase Replicates the Ends of Chromosomes
  • Telomere Length Is Regulated by Cells and
    Organisms

35
Telomerase is a Reverse transcriptase that
carries its own RNA template
36
Telomerase solves the problem of incomplete
lagging strand synthesis
37
Telomeres are sequestered in special chromatin
structures
38
Many Somatic Cells have low telomerase atctivity,
Some cancer cells have enhanced acivity
39
DNA REPAIR
  • Without DNA Repair, Spontaneous DNA Damage Would
    Rapidly Change DNA Sequences
  • The DNA Double Helix Is Readily Repaired
  • DNA Damage Can Be Removed by More Than One
    Pathway
  • The Chemistry of the DNA Bases Facilitates Damage
    Detection
  • Double-Strand Breaks are Efficiently Repaired
  • Cells Can Produce DNA Repair Enzymes in Response
    to DNA Damage
  • DNA Damage Delays Progression of the Cell Cycle

40
Nucleotides in DNA are susceptible to many types
of damage
41
Depurination and Cytosine Deamination
42
Replication fixes mutations on one strand
43
Thymine dimers can be formed after UV irradiation
44
Excision repair
45
Double-strand breaks are the most serious lesions
46
GENERAL RECOMBINATION
  • General Recombination Is Guided by Base-pairing
    Interactions Between Two Homologous DNA Molecules
  • Meiotic Recombination Is Initiated by
    Double-strand DNA Breaks
  • DNA Hybridization Reactions Provide a Simple
    Model for the Basepairing Step in General
    Recombination
  • The RecA Protein and its Homologs Enable a DNA
    Single Strand to Pair with a Homologous Region of
    DNA Double Helix
  • There Are Multiple Homologs of the RecA Protein
    in Eucaryotes, Each Specialized for a Specific
    Function
  • General Recombination Often Involves a Holliday
    Junction
  • General Recombination Can Cause Gene Conversion
  • General Recombination Events Have Different
    Preferred Outcomes in Mitotic and Meiotic Cells
  • Mismatch Proofreading Prevents Promiscuous
    Recombination Between Two Poorly Matched DNA
    Sequences

47
Homologous recombination repairs spontaneous
breaks in DNA and those induced in Meiotic
crossing-over
48
Synapsis Alignment of complementary DNA strands
by base pairing
49
Structure and Mechanism of the RecA protein from
E. coli
50
Robin Hollidays Model for a double strand
crossover junction
51
RuvA,B and C can catalyze branch migration,
mobilize holiday junctions
52
Resolution of Meiotic Crossovers
53
Recombination between repeated sequences can lead
to deletions and inversions Mismatch detection
minimizes aberrant recombination
54
SITE-SPECIFIC RECOMBINATION
  • Mobile Genetic Elements Can Move by Either
    Transpositional or Conservative Mechanisms
  • Transpositional Site-specific Recombination Can
    Insert a DNA Element into Any DNA Sequence
  • DNA-only Transposons Move By DNA Breakage and
    Joining Reactions
  • Some Viruses Use Transpositional Site-specific
    Recombination to Move Themselves into Host Cell
    Chromosomes
  • Retroviral-like Retrotransposons Resemble
    Retroviruses, but Lack a Protein Coat
  • A Large Fraction of the Human Genome Is Composed
    of Nonretroviral Retrotransposons
  • Different Transposable Elements Predominate in
    Different Organisms
  • Genome Sequences Reveal the Approximate Times
    when Transposable Elements Have Moved
  • Conservative Site-specific Recombination Can
    Reversibly Rearrange DNA
  • Conservative Site-Specific Recombination Can be
    Used to Turn Genes On or Off

55
Mobile DNA elements in bacteria
  • Insertion sequences
  • Cis acting DNA sites - end sequences (red)
  • Trans-acting Protein factors - transposase (blue)
  • Transposons are compound elements

56
Transposition Mechanisms Replicative and non
replicative
57
RNA elements retroviruses and retrotransposons
58
Repeat orientation determines recombination
outcome
59
Lambda Life Cycle
  • coordinated transcription and site specific
    recombination

60
Site-specific recombination by l integrase
61
Bacteriophage Mu
  • Transposase is encoded by A and B genes
  • C represses expression of (almost) all other
    genes
  • Gin is a site specific recombinase

62
Mu replicates by transposition
  • The Mu C protein (AKA Repressor, Rep) is
    analogous to the lambda repressor
  • TS mutants repress at the permissive temperature
    but fail to function at higher temperatures.
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