1. DNA metabolism Replication Early on - “Template” so molecules can line up in a specific order and be joined to create a new macromolecule 1940s - DNA = genetic material 1950s - structure identified how it could act as a template for replication and transmission of genetic info One strand is the complement of the other

2. DNA metabolism Replication Rules Replication is 1. semi-conservative 2. bidirectional (Leading & Lagging strand synthesis) 3. Synthesized by polymerases 4. Highly accurate (proofreading)

3. DNA metabolism Replication Rules Replication 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 ( 15 N) (b) Transfer DNA to medium with only light N ( 14 N), after 1 gen (c) Transfer DNA to medium with only light N ( 14 N), after 2 gen

4. DNA metabolism Replication Rules Are parental strands completely unwound before replication? Yes Does replication proceed in one direction or both? Cairns

5. DNA metabolism Replication Rules Does replication begin at a unique point? Yes, called origin

6. DNA metabolism Replication Rules DNA synthesis proceeds 5’  3’ and is semidiscontinuous How can both strands be synthesized simultaneously? 1 strand synthesized in short fragments bidirectional

7. DNA metabolism Replication Rules DNA is synthesized by DNA polymerases DNA 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

8. DNA metabolism Replication Rules Accuracy of replication High fidelity E.Coli, 1 mistake/10 9 to 10 10 nts added E.Coli chromosome (~10 6 ), so error occurs once every 1000 to 10,000 replications Discrimination between correct and incorrect nts relies on H-bonding between correct pairs and geometry of AT and GC bp

9. DNA metabolism Replication Rules Accuracy of replication Proofreading for mistakes 3’  5’ exonuclease activity double checks each nt after it is added

10. DNA metabolism Stages of Replication Initiation Only phase that is regulated so that replication occurs only once every cell cycle

11. DNA metabolism Stages of Replication Elongation

12. DNA metabolism Stages of Replication Elongation RNA primers removed by DNA pol I (5’  3’ exo)

13. DNA metabolism Stages of Replication Elongation

14. DNA metabolism Stages of Replication Termination Ter sequences bound by Tus (terminus utilization substance) Ter-Tus halts fork

15. DNA metabolism DNA Replication Much more complicated in eukaryotes Lots more proteins Linear chromosomes (how replicate very ends?)

16. DNA metabolism DNA Repair DNA damage from: 1. spontaneous loss of exocyclic amino group (deamination) C  U occurs once every 10 7 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 10 5 purines in a day (10,000x a day) Slower for pyrimidines 3. UV damage causes pyrimidine dimers 4. Reactive chemicals Nitrous acid precursors Alkylating agents (nitrogen mustard, DMS, SAM) 5. Oxidative Damage H 2 O 2, OH, O 2 -

17. DNA Mismatch Repair Correction of mismatches increases fidelity by 100 to 1000-fold DNA metabolism Repairs mismatches up to 1000 bp from hemi- methylated GATC

18. DNA Repair Defects in genes encoding proteins involved in mismatch repair, nucleotide-excision repair, and recombinational repair can cause cancer Nucleotide-excision repair sole 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 repair Hereditary nonpolyposis colon cancer (HNPCC) linked to defects in these genes Recombinational repair Recombination - 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 BRCA2 Women with defects in these genes have a >80% chance of developing breast cancer DNA metabolism

19. Stages of Replication Elongation