1. DNA Replication Lecture 11 Fall 2008

2. Read pgs. 305-312

3. Nucleic Acid Structure Deoxyribonucleic Acid (DNA) –Double strand Ribonucleic Acid (RNA) –Single strand Nucleic Acid: long chain of nucleotides 3 components of nucleotides 5 carbon sugar Phosphate group (PO 4 - ) Nitrogenous bases –Nucleoside = sugar + base (no phosphate group) 1 Fig. 5.27

4. Nucleic Acid Structure Nitrogenous bases Pyrimidines 6-membered ring of carbon and nitrogen –Cytosine (C) –Thymine (T) –Uracil (U) replaces T in RNA Purines 6-membered ring fused to a 5-membered ring –Adenine (A) –Guanine (G) 2 Fig. 5.27

5. Nucleic Acid Structure 5 carbon sugar –Ribose in RNA –Deoxyribose in DNA Missing oxygen at 2’ Carbons numbered 1 to 5 – prime’ 3

6. DNA Structure Long chain of nucleotides –Allows for unique arrangement of 4 bases Sugar-phosphate backbone –Phosphodiester linkage Covalent bond between sugar group of one nucleotide and phosphate group of another nucleotide 5’ end with phosphate group 3’ end with hydroxyl group (OH) Fig. 5.27 4

7. DNA Structure DNA molecule Double helix –Two strands Antiparallel –Strands oriented in opposite directions Complementary base pairing –T + A –C + G Hydrogen bonds between the base pairs Van der Waals interactions between stacked bases Fig. 16.27 5

8. DNA Replication Cell division requires the duplication of genetic material DNA is a template –Two strands separate –Free nucleotides bond to template and form “daughter” DNA strand 6 See Fig. 5.28

9. DNA Replication Origins of replications –Short stretches of DNA with specific nucleotide sequence –DNA separates, forming replication bubble –Replication continues in both directions until completed –Prokaryotes One origin of replication See Fig. 16.12 7

10. DNA Replication Origins of replications –Eukaryotes Many origins (100s to 1000s) Replication bubbles eventually fuse –Replication fork Y-shaped region where parental DNA strand is unwound into 2 single strands Fig. 16.12 8

11. DNA Replication How does DNA separate? Helicases –Unwinds and separates DNA strands –Catalyzes breaking of hydrogen bonds between nucleotides Single-strand binding proteins –Stabilizes separated strands Topoisomerase –Releases strain on unwinding DNA –Cuts, twists and rejoins DNA downstream of replication fork Fig. 16.13 9

12. DNA Replication How is DNA synthesis initiated? –Primase Adds a primer - short section of RNA –5-10 nucleotides long Necessary because DNA polymerases can only add nucleotides to an existing chain Fig. 16.13 10

13. DNA Replication DNA polymerases –Catalyze synthesis of new DNA by adding nucleotides to preexisting chain –Prokaryotes DNA polymerase III & DNA polymerase I –Eukaryotes ~ 11 DNA polymerases identified 11

14. DNA Replication Nucleoside triphosphate –Sugar, base + 3 phosphate groups –Removal of 2 phosphates catalyzed by DNA polymerase III Nucleotides can only be added at 3’ end Elongation in 5’ to 3’ direction Fig. 16.14 12

15. Leading and Lagging Strands Leading strand –The new complementary DNA strand synthesized continuously along the template strand toward the replication fork –DNA polymerase III and sliding clamp Fig. 16.15 13

16. Leading and Lagging Strands Lagging strand A discontinuously synthesized DNA strand that elongated by means of Okazaki fragments –A short segment of DNA synthesized away from the replication fork 100-200 nucleotides (eukaryotes) Requires multiple primers Fig. 16.16 14

17. Leading and Lagging Strands DNA polymerase I –Replaces RNA nucleotides of primer with DNA nucleotides DNA ligase –Joins the Okazaki fragments Fig. 16.16 15

18. DNA Repair Errors in completed DNA molecule –1 in 10 billion nucleotides Initial pairing errors in DNA replication –1 in 100,000 nucleotides Corrections during replication –DNA polymerase proofreads If error in match, nucleotide removed and replaced –Mismatch repair Repair by other enzymes if DNA polymerase missed the error 16

19. DNA Repair Corrections after replication –~100 repair enzymes identified in in E. coli –~130 repair enzymes identified in humans Nucleotide excision repair –E.g., repair of thymine dimers Covalent linking of adjacent thymine bases Causes DNA to buckle Caused by UV radiation –Nuclease cuts damaged DNA at two points –DNA polymerase adds nucleotides –DNA ligase joins nucleotides Fig. 16.18 17

20. DNA Repair Replicating ends of linear DNA molecules Nucleotides can only be added at 3’ end of existing strand No way to replace the primer on the 5’ end Linear DNA molecules grow shorter with each replication –In somatic cells Fig. 16.19 18

21. DNA Repair Telomeres –Repeating sequence of nucleotides at ends of linear chromosomes TTAGGG in humans Repeated 100 to 1000 times –Do not contain genes –Chromosomes continue to shorten –Cell eventually dies 19

22. DNA Repair Preserving DNA ends in meiosis Telomerase –Catalyzes lengthening of telomeres in germ cells –Preserves length of chromosomes in gametes –Not active in most somatic cells 20

23. DNA Repair Telomerase and cancer –Chromosomes of somatic cells gradually shorten Telomere loss signals cells to enter non-dividing stage –If telomerase activated in somatic cells, cell may continue to divide May become cancerous 21