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DNA metabolismReplicationEarly on - “Template” so molecules can line up in a specific order and be joined to create a new macromolecule1940s - DNA = genetic material1950s - structure identified how it could act as a template for replication and transmission of genetic info
One strand is the complement of the other
DNA metabolismReplication RulesReplication is 1. semi-conservative2. bidirectional (Leading & Lagging strand synthesis)3. Synthesized by polymerases4. Highly accurate (proofreading)
DNA metabolismReplication RulesReplication is semi-conservative (each DNA strand serves as a template for the synthesis of a new strand, producing 2 new DNA molecules, each with one new strand and one old strand)
1957 - Meselson-Stahl(a) Grow DNA for many generations in medium with heavy N (15N)(b) Transfer DNA to medium with only light N (14N), after 1 gen(c) Transfer DNA to medium with only light N (14N), after 2 gen
DNA metabolismReplication RulesAre parental strands completely unwound before replication? YesDoes replication proceed in one direction or both?
Cairns
DNA metabolism
Replication RulesDNA synthesis proceeds 5’3’ and is semidiscontinuousHow can both strands be synthesized simultaneously? 1 strand synthesized in short fragments
bidirectional
DNA metabolismReplication RulesDNA is synthesized by DNA polymerasesDNA Polymerase requires
1. template (bp rules)2. primer (short strand with free 3’-OH)
1955 - Kornberg purified and characterized DNA polymerase I from E.Coli
DNA metabolismReplication RulesAccuracy of replicationHigh fidelityE.Coli, 1 mistake/109 to 1010 nts addedE.Coli chromosome (~106), so error occurs once every 1000 to 10,000 replicationsDiscrimination between correct and incorrect nts relies on H-bonding between correct pairs and geometry of AT and GC bp
DNA metabolismReplication RulesAccuracy of replicationProofreading for mistakes
3’5’ exonuclease activity double checks each nt after it is added
DNA metabolismStages of ReplicationInitiationOnly phase that is regulated so that replication occurs only once every cell cycle
DNA metabolismStages of ReplicationTerminationTer sequences bound by Tus (terminus utilization substance)Ter-Tus halts fork
DNA metabolism
DNA ReplicationMuch more complicated in eukaryotesLots more proteinsLinear chromosomes (how replicate very ends?)
DNA metabolism
DNA RepairDNA damage from:1. spontaneous loss of exocyclic amino group (deamination)
C U occurs once every 107 C residues in a day (100x a day)A G occurs 100x slower
2. Hydrolysis of bond between sugar and base (apurinic residue)Occurs once every 105 purines in a day (10,000x a day) Slower for pyrimidines
3. UV damage causes pyrimidine dimers
4. Reactive chemicalsNitrous acid precursorsAlkylating agents (nitrogen mustard, DMS, SAM)
5. Oxidative DamageH2O2, •OH, •O2
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DNA Mismatch RepairCorrection of mismatches increases fidelity by 100 to 1000-fold
DNA metabolism
Repairs mismatches up to 1000 bp from hemi-methylated GATC
DNA Repair
Defects in genes encoding proteins involved in mismatch repair, nucleotide-excision repair, and recombinational repair can cause cancer
Nucleotide-excision repairsole repair pathway for pyrimidine dimers
genetic defect causes XP, xeroderma pigmentosa, these individuals are extremely sensitive to sunlight and quickly develop sunlight-induced skin cancer
Mismatch repairHereditary nonpolyposis colon cancer (HNPCC) linked to defects in these genes
Recombinational repairRecombination - linear sequence of DNA altered by cleavage and rejoining of chromosome (involves RecA protein)
Repair of this type sometimes needed to reconstruct replication fork
Human breast cancer genes (BRCA1 and BRCA2) produce proteins that interact with the human homolog of RecA, therefore these are linked to recombination repair
10% of breast cancers have defects in BRCA1 or BRCA2Women with defects in these genes have a >80% chance of developing breast cancer
DNA metabolism