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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 • 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 • 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 • 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 • 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
The DNA Replication Fork Is Asymmetrical • Leading and lagging strands have different requirements
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
The eucaryotic fork is like the procaryotic but with more specialization
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”
A Strand-directed Mismatch Repair System Removes Replication Errors That Escape from the Replication Fork
Type 2 topoisomerase nicks both strands - Unwinds and Untangles - Makes DNA “ethereal”
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
Initiation and mismatch repair in bacteria are both controlled by methylation
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 • 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
A Large Multisubunit Complex Binds to Eucaryotic Origins of Replication
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
Telomerase solves the problem of incomplete lagging strand synthesis
Many Somatic Cells have low telomerase atctivity, Some cancer cells have enhanced acivity
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
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
Homologous recombination repairs spontaneous breaks in DNA and those induced in Meiotic crossing-over
Synapsis:Alignment of complementary DNA strands by base pairing
Robin Holliday’s Model for a double strand crossover junction