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The initiation of yeast DNA replication Questions? Contact michael.weinreich@vai

BMB801 Lecture 16 -- Dr. Michael Weinreich 10/4/06. The initiation of yeast DNA replication Questions? Contact michael.weinreich@vai.org Van Andel Research Institute Grand Rapids, MI 49503. Initiation of DNA Replication.

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The initiation of yeast DNA replication Questions? Contact michael.weinreich@vai

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  1. BMB801 Lecture 16 -- Dr. Michael Weinreich 10/4/06 The initiation of yeast DNA replication Questions? Contact michael.weinreich@vai.org Van Andel Research Institute Grand Rapids, MI 49503

  2. Initiation of DNA Replication Essential for cell growth, development, integrity of the genetic information Each origin initiates replication only once per cell cycle Highly regulated. Commitment to DNA replication is an irreversible decision Many initiation proteins are upregulated in cancer cells

  3. Initiation of DNA Replication Essential for cell growth, development, integrity of the genetic information Each origin initiates replication only once per cell cycle Highly regulated. Commitment to DNA replication is an irreversible decision Many initiation proteins are upregulated in cancer cells QUESTIONS What are the requirements for the initiation of DNA replication? How is initiation limited to once per cell cycle? What effect might chromatin structure have on initiation? Overview of the temporal nature of replication initiation

  4. DNA Replication Begins at Specific Sequences Fiber Autoradiography Visualize 3H-Thymidine incorporation following a short pulse

  5. Isolation of Autonomously Replicating Sequences origin Restriction digest of yeast chromosomal DNA “Shotgun” clone into a URA3 origin-less plasmid Transform into ura3 yeast Select for URA+ cells

  6. Isolation of Autonomously Replicating Sequences EcoRI URA3 URA3 URA3 EcoRI URA+

  7. Isolation of Autonomously Replicating Sequences EcoRI URA3 URA3 URA3 EcoRI

  8. Isolation of Autonomously Replicating Sequences EcoRI URA3 URA3 URA3 EcoRI Ura-

  9. Isolation of Autonomously Replicating Sequences EcoRI URA3 URA3 URA3 EcoRI URA+ -URA plate

  10. { A B ARS elements consist of two domains ARS element ~150-200bp

  11. { A B ARS elements share a common sequence - the ACS ARS element ~150-200bp WTTTAYRTTTW ARS Core Consensus Sequence W= A or T R= A or G Y= T or C

  12. { A B Plasmid stability measurements of linker scan mutants ARS element “GGTCGAC” ~150-200bp SalI 1. Transform plasmid into yeast selecting for URA3 marker Failure to recover high frequency of transformation (HFT) - essential sequences 2. Grow yeast in medium lacking uracil to get a population of cells 3. Grow in medium containing uracil to allow plasmid loss events 4. Calculate percentage of cells containing plasmid after X generations

  13. Linker scan analysis of ARS315 B1 A B2 IS Percentage of plasmid containing cells _ _ HFT + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ACS

  14. Linker scan analysis reveals modular structure of origins A B1 B2 B3 ARS1 ARS307 ACS Important Essential

  15. There are at least two broad classes of origins in S. cerevisiae A B1 B2 B3 ARS1 A B1 B2 IS ARS315 ARS305 ARS307 ? ACS Inhibitory element

  16. Jacob and Brenner’s Replicon Model Initiator protein Replicator WTTTAYRTTTW Jacob, F., and S. Brenner. 1963. [On the regulation of DNA synthesis in bacteria: the hypothesis of the replicon.] C R Hebd Seances Acad Sci 256: 298-300.

  17. Jacob and Brenner’s Replicon Model Initiator protein -- DnaA Replicator -- OriC 13mers DnaA boxes E. coli

  18. Jacob and Brenner’s Replicon Model Initiator protein -- ORC (Origin Recognition Complex) A B1 B2 B3 Replicator -- ARS Bell, SP and Stillman, B. (1992) ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex. Nature 357(6374):128-34.

  19. ORC binds to origins of replication ORC A B1 B2 origin -ORC is a six-subunit protein complex that binds to ARS elements. -Specific DNA binding requires ATP -All subunits are essential and are required for initiation -ORC binds ARS elements throughout the cell cycle

  20. …and then recruits Cdc6p and Cdt1p during early G1 Cdc6p Cdt1p ORC ORC A B1 B2 A B1 B2 origin

  21. Cdc6 is a AAA+ protein required for pre-RC assembly K114A mutant is ts: cdc6-4

  22. The “pre-Replicative Complex” assembles at all origins in G1 MCM ORC A B1 B2 pre-RC

  23. Kinases then activate the initiation of DNA synthesis MCM ORC ORC A B1 B2 A B1 B2 Cdc45p GINS pre-RC Cdc7p-Dbf4p + Cdk1-Clb Ser/Thr protein kinases

  24. Origin unwinding occurs MCM ORC A B1 B2 Cdc45p GINS

  25. Pol a -primase MCM ORC A B1 B2 Cdc45p GINS … followed by the assembly of DNA polymerases and bi-directional DNA synthesis

  26. P Cdt1 P Cdk Kinase

  27. How do cyclin-dependent kinases prevent pre-RC formation? CyclinB-Cdks phosphorylate ORC, Cdc6p and MCM proteins ORC phosphorylation inhibits its activity through an unknown mechanism Cdc6p phosphorylation causes is proteolysis MCM phosphorylation excludes it from the nucleus, as well as Cdt1p P P P MCM ORC ORC A B1 B2 A B1 B2 High Cdk levels pre-RC Low Cdk levels

  28. P Cdt1 P Cdk Kinase

  29. ORC and Abf1p position nucleosomes outside ARS1 Abf1p ORC A B1 B2 B3

  30. … facilitating pre-RC formation at the origin Cdc6p Cdt1p Abf1p MCM ORC A B1 B2 B3

  31. Origins lacking a B3 element may be sensitive to nucleosome intrusion Cdc6p Cdt1p Abf1p MCM ORC A B1 B2 B3 ORC A B1 B2

  32. …that inhibits pre-RC assembly Cdc6p Cdt1p Abf1p MCM ORC A B1 B2 B3 Cdc6p Cdt1p ORC A B1 B2

  33. IS element may position a nucleosome over origin A B1 B2 B3 ARS1 A B1 B2 IS ARS315 nucleosome ARS305 ARS307 ? ACS

  34. Position of ARS elements along chromosome III 302 303 320 314 318 301 308 300 304 305 306 309 315 310 313 316 317 319 307 Chromosome III < 10% Inactive < 10%

  35. Position of ARS elements along chromosome III Early origins 302 303 320 314 318 301 308 300 304 305 306 309 315 310 313 316 317 319 307 Chromosome III < 10% Inactive < 10%

  36. Position of ARS elements along chromosome III Early origins 302 303 320 314 318 301 308 300 304 305 306 309 315 310 313 316 317 319 307 Chromosome III < 10% Inactive < 10% Late replicating Telomeres

  37. Replication timing along chromosome VI Raghuraman MK et al. (2001) Replication dynamics of the yeast genome Science 294(5540):115-21.

  38. BMB801 Lecture 17 -- Dr. Michael Weinreich 10/6/06 DNA damage during S-phase, telomeres, late replication and the centromere Questions? Contact michael.weinreich@vai.org Van Andel Research Institute Grand Rapids, MI 49503

  39. Linker scan analysis reveals modular structure of origins A B1 B2 B3 ARS1 ARS307 ACS Important Essential

  40. { A B Plasmid stability measurements of linker scan mutants ARS element ~150-200bp “Linker scans” are typically ~6-10bp in length. They change the existing sequence at multiple base pairs without the adding or deleting nucleotides and introduce a novel restriction site. For example, an 8bp XhoI linker scan across this region might change the existing sequence to CCTCGAGG at each position above.

  41. Formation of the pre-RC occurs during G1 Cdc6p MCM Cdt1p ORC ORC ORC A B1 B2 A B1 B2 A B1 B2 origin ORC, Origin Recognition Complex MCM, DNA helicase Cdc6p and Cdt1p, helicase loaders

  42. Kinases then activate the initiation of DNA synthesis MCM ORC ORC A B1 B2 A B1 B2 Cdc45p GINS pre-RC Cdc7p-Dbf4p + Cdk1-Clb Ser/Thr protein kinases

  43. Pol a -primase MCM ORC A B1 B2 Cdc45p GINS … by promoting the assembly of DNA polymerases and bi-directional DNA synthesis

  44. Late replication, the telomere and functions of the centromere QUESTIONS 5. What determines the temporal order of replication during S-phase? 6. How might DNA damage affect DNA replication? 7. What are telomeres and how are they replicated? 8. Overview of the centromere and kinetochore

  45. The “pre-Replicative Complex” assembles at all origins in G1 MCM ORC Early and late origins assemble the pre-RC during G1 Since some origins are not activated until late S-phase, the regulatory step must occur after pre-RC formation Cdc7-Dbf4 and Cdk1-Clb5,6? A B1 B2 pre-RC

  46. Replication timing along chromosome VI Raghuraman MK et al. (2001) Replication dynamics of the yeast genome Science 294(5540):115-21.

  47. MMS activates the intra-S-phase checkpoint which inhibits late origin firing DNA damage (MMS) Rad9p Mec1p (ATR) Mec2/Ddc1 (ATRIP) Chk1p Rad53p (Chk2) Inhibit late origin firing G2/M origin WT

  48. MMS activates the intra-S-phase checkpoint which inhibits late origin firing DNA damage (MMS) Rad9p Mec1p (ATR) Mec2/Ddc1 (ATRIP) Chk1p Rad53p (Chk2) Inhibit late origin firing G2/M origin WT +MMS Slows S-phase

  49. Pol a -primase MCM ORC Cdc45p A B1 B2 GINS Cdc7p-Dbf4p Dbf4p is phosphorylated following replication arrest P Dbf4p-Cdc7p ? pre-RC Late origins Rad53p Mec1p DNA damage Replication arrest

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