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 RulesDoes replication begin at a unique point? Yes, called origin

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 ReplicationElongation

DNA metabolismStages of ReplicationElongationRNA primers removed by DNA pol I (5’→3’ exo)

DNA metabolismStages of ReplicationElongation

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

DNA metabolismStages of ReplicationElongation