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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. THE MAINTENANCE OF DNA SEQUENCES.

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CHAPTER 5 DNA REPLICATION, REPAIR AND RECOMBINATION

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Chapter 5 dna replication repair and recombination l.jpg

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


The maintenance of dna sequences l.jpg

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


Mutation rates are relatively constant but proteins evolve at different rates l.jpg

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


Dna replication mechanisms l.jpg

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


Base pairing underlies dna replication and dna repair l.jpg

Base-pairing Underlies DNA Replication and DNA Repair


Dna replication is semiconservative l.jpg

DNA replication is semiconservative


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Fidelity of DNA Replication Requires Proofreading


Many polymerases contain separate 3 5 editing domains l.jpg

Many polymerases contain separate 3’->5’ editing domains


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The DNA Replication Fork Is Asymmetrical

  • Leading and lagging strands have different requirements


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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


Dna primase synthesizes short rna primer molecules on the lagging strand l.jpg

DNA Primase Synthesizes Short RNA Primer Molecules on the Lagging Strand


Helicases open up the dna double helix l.jpg

Helicases - Open Up the DNA Double Helix


Single strand binding proteins keep ssdna out of trouble l.jpg

Single strand binding proteins keep ssDNA out of trouble


Clamp subunits tether a moving dna polymerase to the dna l.jpg

Clamp subunits tether A Moving DNA Polymerase to the DNA


Clamp loading and unloading on the lagging strand l.jpg

Clamp loading and unloading on the lagging strand


The replication machine expanded l.jpg

The replication machine - expanded


The replication machine trombone model l.jpg

The replication machine - trombone model


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The eucaryotic fork is like the procaryotic but with more specialization


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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”


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A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Fork


Dna topoisomerase prevents dna tangling during replication l.jpg

DNA Topoisomerase Prevents DNA Tangling During Replication


Type 1 topoisomerase nicks only one strand unwinds only l.jpg

Type 1 topoisomerase nicks only one strand - unwinds only


Type 2 topoisomerase nicks both strands unwinds and untangles makes dna ethereal l.jpg

Type 2 topoisomerase nicks both strands - Unwinds and Untangles - Makes DNA “ethereal”


The initiation of dna replication l.jpg

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


Dna synthesis begins at replication origins l.jpg

DNA Synthesis Begins at Replication Origins


Bacteriahave a single origin l.jpg

BacteriaHave a Single Origin


Initiation and mismatch repair in bacteria are both controlled by methylation l.jpg

Initiation and mismatch repair in bacteria are both controlled by methylation


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Origins can be mapped by pulse labeling


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Different DNA regions have distinct timings of replication during the S phase of the eukaryotic cell cycle


Well defined dna sequences serve as replication origins in a simple eucaryote the budding yeast l.jpg

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


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Yeast Origins have been identified by genetic means


A large multisubunit complex binds to eucaryotic origins of replication l.jpg

A Large Multisubunit Complex Binds to Eucaryotic Origins of Replication


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Chromatin is assembled following replication forks


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COMPLETION OF DNA REPLICATION IN CHROMOSOMES

  • Telomerase Replicates the Ends of Chromosomes

  • Telomere Length Is Regulated by Cells and Organisms


Telomerase is a reverse transcriptase that carries its own rna template l.jpg

Telomerase is a Reverse transcriptase that carries its own RNA template


Telomerase solves the problem of incomplete lagging strand synthesis l.jpg

Telomerase solves the problem of incomplete lagging strand synthesis


Telomeres are sequestered in special chromatin structures l.jpg

Telomeres are sequestered in special chromatin structures


Many somatic cells have low telomerase atctivity some cancer cells have enhanced acivity l.jpg

Many Somatic Cells have low telomerase atctivity, Some cancer cells have enhanced acivity


Dna repair l.jpg

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


Nucleotides in dna are susceptible to many types of damage l.jpg

Nucleotides in DNA are susceptible to many types of damage


Depurination and cytosine deamination l.jpg

Depurination and Cytosine Deamination


Replication fixes mutations on one strand l.jpg

Replication “fixes” mutations on one strand


Thymine dimers can be formed after uv irradiation l.jpg

Thymine dimers can be formed after UV irradiation


Excision repair l.jpg

Excision repair


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Double-strand breaks are the most serious lesions


General recombination l.jpg

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


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Homologous recombination repairs spontaneous breaks in DNA and those induced in Meiotic crossing-over


Synapsis alignment of complementary dna strands by base pairing l.jpg

Synapsis:Alignment of complementary DNA strands by base pairing


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Structure and Mechanism of the RecA protein from E. coli


Robin holliday s model for a double strand crossover junction l.jpg

Robin Holliday’s Model for a double strand crossover junction


Ruva b and c can catalyze branch migration mobilize holiday junctions l.jpg

RuvA,B and C can catalyze branch migration, mobilize holiday junctions


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Resolution of Meiotic Crossovers


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Recombination between repeated sequences can lead to deletions and inversions: Mismatch detection minimizes aberrant recombination


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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


Mobile dna elements in bacteria l.jpg

Mobile DNA elements in bacteria

Insertion sequences

Cis acting DNA sites - end sequences (red)

Trans-acting Protein factors - transposase (blue)

Transposons are compound elements


Transposition mechanisms replicative and non replicative l.jpg

Transposition Mechanisms:Replicative and non replicative


Rna elements retroviruses and retrotransposons l.jpg

RNA elements : retroviruses and retrotransposons


Repeat orientation determines recombination outcome l.jpg

Repeat orientation determines recombination outcome


Lambda life cycle l.jpg

Lambda Life Cycle

coordinated transcription and site specific recombination


Site specific recombination by l integrase l.jpg

Site-specific recombination by l integrase


Bacteriophage mu l.jpg

Bacteriophage Mu

Transposase is encoded by A and B genes

C represses expression of (almost) all other genes

Gin is a site specific recombinase


Mu replicates by transposition l.jpg

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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