* The position on the DNA at which replication start points are found. (4 methods discussed)

1. Characteristics used to define Origins of Replication * The position on the DNA at which replication start points are found. (4 methods discussed) * A DNA sequence that when added to a non-replicating DNA causes it to replicate. * A DNA sequence whose mutation abolishes replication. * A DNA sequence that in vitro is the binding target for enzyme complexes known to function in initiation of DNA replication.

2. 1) In synchronously replicating cells, density transfer experiments can reveal which sequences replicate earliest. What is the direction of fork movement here?

3. TR50 Calculation for human chromosomal probes on genome tiling arrays (ENCODE project) TR50 - Time at which 50% of the locus is replicated In the example below, ‘probe A’ has a TR50 of 1.25hr (80% at 2hr, 0% at 0hr) ‘probe B’ has a TR50 of 6.33hr (100% at 8hr, 40% at 6hr) Example: Probe Probe

4. Predicted origins, UCSC genome browser, ENCODE: UVa DNA replication tracks Neerja Karnani Chris Taylor Ankit Malhotra

5. 2) Electron microscopy of bubbles after restriction enzyme digestion

7. 3) Hybridizing lagging strand nascent DNA to strand-specific fragments shows site of switch from leading to lagging strand synthesis A B C D E F G H Autorad after hybridizing nascent strands DNA spotted on membrane

8. E F E F A B C D A B C D G H G H 3b) Hybridizing of leading strand nascent DNA shows sites near origins that are enriched in nascent strand G H Probes A B C D E F Okazaki fragments: <500 bases long Leading strand nascent strands: > 500 bases long Leading strand nascent strands near origins < 2000 bases long Nascent DNA 500-2000 base long used to probe fragments along chromosome Nascent strand abundance Nascent strand peak at origin DNA spotted on array

9. 4) 2D Gel electrophoresis of replication intermediates followed by hybridization with a DNA fragment reveals whether a replication bubble originates in the fragment Dimension 1: separates by size; dimension 2: separates by shape

10. Sequence that when added to a non-replicating DNA causes it to replicate; this approach was used to identify Autonomously Replicating Sequences (ARS) in yeast.

11. Grow under non-selective conditions Sequence that when mutated causes a replicating DNA to fail to replicate; this approach was used to identify essential elements in an (ARS) in yeast Yeast containing Leu2 on a plasmid Plate yeast on Leucine minus plates to estimate rate of plasmid loss

12. Linker scanning mutations in yeast ARS1 Examples of mutants WT-ARS1 B3 B2 B1 A % URA+ colonies

14. OriC in E. coli chromosomal DNA

15. Initiation of DNA replication in E. coli

16. Nature Structural & Molecular Biology – 13, 676 - 683 (2006) Berger JM

17. DnaA and oriC independent replication initiation in E. coli from hairpins, D-loops and R loops Two primosomes for E. coli chromosomal replication Site of primosome assembly oriC, A-site n'-pas, D-loop, R-loop Recognition DnaA protein PriA protein Auxilliary proteins for (HU for oriC) PriB loading helicase PriC DnaT Helicase DnaB/DnaC Priming Primase

18. SV40: a polyomavirus that is very useful for studying eukaryotic replication enzymes

19. Early genes : proteins essential for viral DNA replication

20. T antigen binding sites cluster around the ori

21. Properties of SV40 T antigen * 98% nuclear: NLS * origin-specific DNA binding * DNA independent ATPase * ATP dependent DNA helicase * Binds to DNA polymerases, AP2, p53, Rb * Undergoes phosphorylation etc.

22. Li & Kelly SV40 DNA replication In vitro. PNAS (1984) 81:6973 What else could they do to validate their system?

23. Fractionation to homogeneity

24. ELONGATION FACTORS ARE CONSERVED

30. RF-C RF-C RF-C is a five-subunit complex All subunits are related in sequence and have ATP binding motifs ATP hydrolysis by RF-C is associated with the loading of PCNA RF-C is the functional homolog of the clamp-loader g complex

32. Polymerase switching occurs even on lagging strands; pol d does most of DNA synthesis

33. Dna2 endonuclease is also necessary for this step

34. How do you think the PCNA is removed after ligation?

36. PCNA interacts with RF-C, pol d, Fen1, DNA ligase, CAF1 and MCMT Several of these have a common motif used in the interaction: Q-X-X-L/I/M-X-X-F-F/Y p21/CIP1/WAF1, a protein induced by the tumor suppressor p53 uses the same motif to interact with PCNA What effect is p21 expected to have on DNA replication/repair?

37. FEN1 interacts with PCNA

39. DNA Helicase * Helicase binds ss DNA * Hydrolyzes ATP to move along ssDNA and peel of substrate DNA * Can move 5‘ to 3’ or 3‘to 5’ or in both directions on ssDNA and is classified as such * Dimers or hexamers

40. Helicases are ATP driven molecular motors Conserved helicase motifs: Includes the following for ATP binding and hydrolysis I = Walker A motif GxGxGKT II = Walker B motif FFFDEad

41. Grow under non-selective conditions Mutation in a yeast gene that causes a failure in MiniChromosome Maintenance (MCM genes) Yeast containing Leu2 on a plasmid Plate yeast on Leucine minus plates to estimate rate of plasmid loss

42. Form hexamer Associates with MCM2-7 Newbies of unknown function MCM8 MCM9

43. MCM homolog from M. thermoautotrophicum (an archaebacterium) has helicase activity. What direction?

44. Mt MCM forms double hexamers

45. T antigen helicase domain: XS Chen, Nature, 2003;423:512-8

46. MtMCM-N terminal (non-helicase) domain: XS Chen, Nature Str. Biol. 2003;10:160-7

47. Model of replicative double-hexameric helicase action : XS Chen, Nature, 2003;423:512-8