1. Replication – copying of DNA The cell invests enormous resources in making sure that replication (copying DNA) is as accurate as possible including elaborate proofreading and repair functions. In comparison, relatively fewer resources are devoted to insuring that transcription (DNA → mRNA) is accurate. Why is replication so important to the cell?

2. Importance of Replication Accurate copying of the genetic information is essential for the viability of new cells Many diseases are the results of mutations in the DNA. Many cancers include types of colon and breast cancer are specifically linked to mutations in proofreading and error repair enzymes of replication. Aging and death (cells will only divide a specific # of times) are linked to changes in DNA. Theory: errors accumulate over time

3. Link to DNA replication animation DNA Interactive Interviews with Arthur Kornberg DNA Replication requires: 1) DNA template 2) Nucleotides triphosphate bases 3) Several classes of proteins including DNA polymerase

4. Fig. 16-12a Origin of replication Parental (template) strand Daughter (new) strand Replication fork Replication bubble Double- stranded DNA molecule Two daughter DNA molecules (a) Origins of replication in E. coli 0.5 µm ORIGIN OF REPLICATION ORIGIN OF REPLICATION – site on DNA at which replication process begins Prokaryotes Single origin of replication Origin is A-T rich ( less energy required to break 2 H-bonds in A-T vs. 3 H- bonds in C-G) Replication is bidirectional Small circular DNA Bidirectional – replication proceeds in both directions outwards from the origin of replication

5. Fig. 16-12b 0.25 µm Origin of replicationDouble-stranded DNA molecule Parental (template) strand Daughter (new) strand Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes Eukaryotes – long linear segments of DNA with multiple origins of replication NOTE :MULTIPLE ORIGINS AND BIDIRECTIONAL REPLICATION Replication Fork – region in which DNA is being actively copied

6. ROLE OF DNA POLYMERASES IN DNA REPLICATION DNA polymerase is actually a complex of many different proteins Main DNA polymerase that elongates strands is called Pol III. DNA Pol I removes RNA primers and replaces RNA with DNA Other DNA polymerases (II,IV and V) are most likely involved in repair.

7. Properties of DNA Polymerase III (major elongation enzyme of replication) DNA polymerase cannot start a strand from scratch; can only add to the length of an existing strand; Cell typically uses RNA as a primer to begin new strands DNA polymerase can only add nucleotides in the 5’ to 3’ direction and requires a single- stranded template to copy DNA polymerase III adds 1000 bp per second to replicating strand

8. Table 16-1

9. Figure 16.15 The main proteins of DNA replication and their functions Link to Harvard site

10. Fig. 16-13 Topoisomerase Helicase Primase Single-strand binding proteins RNA primer 5 5 53 3 3 INITIATION OF REPLICATION – unwinding of DNA and stabilization of single strands HELICASE – UNWINDS DNA SINGLE-STRANDED BINDING PROTEINS – STABLIZES UNWOUND DNA

11. Figure 16.14 Priming DNA synthesis with RNA

12. Figure 16.12 The two strands of DNA are antiparallel

13. Fig. 16-14 A C T G G G GC CC C C A A A T T T New strand 5 end Template strand 3 end 5 end 3 end 5 end 3 end Base Sugar Phosphate Nucleoside triphosphate Pyrophosphate DNA polymerase

14. Remember Replication is 5’ to 3’

15. Fig. 16-16a Overview Origin of replication Leading strand Lagging strand Overall directions of replication 1 2 REPLICATION IS BIDIRECTIONAL – IT PROCEDES OUTWARD FROM ORIGIN IN BOTH DIRECTIONS NOTE TWO REGIONS OF LEADING STRAND SYNTHESIS AND TWO REGIONS OF LAGGING STRAND SYNTHESIS IN REPLICATION BUBBLE

16. Fig. 16-17 Overview Origin of replication Leading strand Lagging strand Overall directions of replication Leading strand Lagging strand Helicase Parental DNA DNA pol III PrimerPrimase DNA ligase DNA pol III DNA pol I Single-strand binding protein 5 3 5 5 5 5 3 3 3 3 1 3 2 4 LEADING STRAND LEADING STRAND– strand which is being elongated in the 5’ → 3’ direction; synthesis is continuous Towards the direction of the replication fork LAGGING STRAND LAGGING STRAND – direction of elongation is in 3’ → 5’ direction; since nucleotides can only be added to 3’ end synthesis is discontinuous away from the direction of the fork.

17. Figure 16.13 Synthesis of leading and lagging strands during DNA replication

18. Fig. 16-15 Leading strand Overview Origin of replication Lagging strand Leading strandLagging strand Primer Overall directions of replication Origin of replication RNA primer “Sliding clamp” DNA poll III Parental DNA 5 3 3 3 3 5 5 5 5 5

19. Fig. 16-16b1 Template strand 5 5 3 3

20. Fig. 16-16b2 Template strand 5 5 3 3 RNA primer 3 5 5 3 1

21. Fig. 16-16b3 Template strand 5 5 3 3 RNA primer 3 5 5 3 1 1 3 3 5 5 Okazaki fragment

22. Fig. 16-16b4 Template strand 5 5 3 3 RNA primer 3 5 5 3 1 1 3 3 5 5 Okazaki fragment 1 2 3 3 5 5

23. Fig. 16-16b5 Template strand 5 5 3 3 RNA primer 3 5 5 3 1 1 3 3 5 5 Okazaki fragment 1 2 3 3 5 5 1 2 3 3 5 5

24. Fig. 16-16b6 Template strand 5 5 3 3 RNA primer 3 5 5 3 1 1 3 3 5 5 Okazaki fragment 1 2 3 3 5 5 1 2 3 3 5 5 1 2 5 5 3 3 Overall direction of replication

25. Ligase Seals Nicks by connecting 5’ monophosphate to 3’ OH

26. Model of the replication fork of phage T7. Hamdan S M et al. PNAS 2005;102:5096-5101 ©2005 by National Academy of Sciences DNA POLYMERASE III SINGLE STRANDED BINDING PROTEINS O.F. = OKAZAKI FRAGMENTS DNA POLYMERASE III

27. Link to Harvard site Link to DNA Replication 2 Mcgraw