DNA REPLICATION: FACTORS THAT INCREASE FIDELITY
• Increase fidelity to 1 mistake per 10^9 bases added
- DNA Pol. proofreading capacity
• DNA Pol. 3′ to 5′ exonuclease activity: can excise/replace incorrect nucleotides
• Prokaryotes: DNA Pol. I (replaces RNA primers) & III (daughter strands) are exonucleases
• Eukaryotes: DNA Pol. delta (lagging strand) & epsilon (leading strand) are exonucleases - Repair mechanisms
Mismatch repair: identifies/fixes replication errors that escape DNA Pol.
• Addresses single-strand breaks in newly replicated DNA
• Cannot repair DNA Damage
MISMATCH REPAIR: E. COLI
• Mismatched nucleotides cannot H-bond –> distorts DNA
• GATC sequence occurs about every thousand nucleotides
• A in GATC is methylated: distinguishes parent strand from daughter strand
• Mut proteins (mismatch repair enzymes in E. Coli)
- MutS recognizes mismatched base and initiates repair by binding
- MutS forms complex with MutL
- MutL binding activates endonuclease MutH
- MutH cleaves daughter strand opposite to adenine-methylation (GATC)
- Exonuclease and helicase excise portion of daughter strand
Intertextual variation exits regarding above step.
- DNA Pol. (III or I) fills gap and ligase seals ends w/ phosphodiester bonds
• DNA Pol synthesizes gap in 5′ to 3′ direction
MISMATCH REPAIR: HUMANS
• MSH proteins: human homologs for Mut proteins (no MutH homolog)
• Daughter strand specificity poorly understood: not adenine-methylation
Theoretical recognition sites: breaks in lagging strand & lengthening 3′ end of lagging strand.
• DNA Pol. (delta and epsilon) fill excised gap
CLINICAL CORRELATION
Lynch Syndrome
• Formerly known as hereditary nonpolyposis colorectal cancer (HNPCC)
• Mutation in human Mut homologs: defective mismatch repair pathway
• Increased risk for colorectal and other cancers
• ~ 3 in every 100 cases of colon cancer caused by Lynch syndrome
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