DNA metabolism - chemistry.creighton.educhemistry.creighton.edu/~jksoukup/lec26.pdfDNA metabolism...
Transcript of DNA metabolism - chemistry.creighton.educhemistry.creighton.edu/~jksoukup/lec26.pdfDNA metabolism...
DNA metabolismReplicationEarly on - “Template” so molecules can line up in a specific order andbe joined to create a new macromolecule1940s - DNA = genetic material1950s - structure identified how it could act as a template forreplication and transmission of genetic info
One strand is the complement of the other
DNA metabolismReplication RulesReplication is1. 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 atemplate for the synthesis of a new strand, producing 2 new DNAmolecules, 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 metabolismReplication RulesDNA synthesis proceeds 5’→3’ and is semidiscontinuousHow can both strands be synthesized simultaneously? 1 strandsynthesized 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 fromE.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 10,000replicationsDiscrimination between correct and incorrect nts relies on H-bondingbetween correct pairs and geometry of AT and GC bp
DNA metabolismReplication RulesAccuracy of replicationProofreading for mistakes
3’→5’ exonuclease activitydouble checks each nt afterit is added
DNA metabolismStages of ReplicationInitiationOnly phase that isregulated so thatreplication occurs onlyonce every cell cycle
DNA metabolismStages of ReplicationTerminationTer sequences bound by Tus(terminus utilizationsubstance)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 to1000 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 areextremely sensitive to sunlight and quickly develop sunlight-inducedskin cancer
Mismatch repairHereditary nonpolyposis colon cancer (HNPCC) linked to defects inthese genes
Recombinational repairRecombination - linear sequence of DNA altered by cleavage andrejoining of chromosome (involves RecA protein)
Repair of this type sometimes needed to reconstruct replication fork
Human breast cancer genes (BRCA1 and BRCA2) produce proteinsthat interact with the human homolog of RecA, therefore these arelinked to recombination repair
10% of breast cancers have defects in BRCA1 or BRCA2Women with defects in these genes have a >80% chance of developingbreast cancer
DNA metabolism