Lecture 3:

1. 5’ 3’ 3’ 5’ 5’ 3’ Lecture 3: Origins and Replication Initiation Analyzing role and function of sequence elements (sequence specific assays) Regulation through feedback inhibition by product 5’ 5’ Lecture 4: Eukaryotic Initiation and Regulation In vivo analysis of protein interactions and complex assembly Increasing the power of genetic tools with better molecular phenotypes

2. Localize factors to origins and/or replication forks Establish order of assembly during initiation and cell cycle progression Develop in vitro system and specific assays Future mechanistic studies (great Bioreg proposals) A Tale of Two Systems E. Coli oriC S. cerevisiaeARS Genetically identify initiation factors Develop in vitro system Establish “purified” system Create partial reactions and structurally analyze intermediates Infer protein function and develop specific assays

3. Prokaryotic and Eukaryotic Replication Initiation Activities 5’ 3’ 5’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ Converting DS DNA to replication fork E. coli S. cerevisiae 1. Recognize initiation site (replication origin) DnaA binds oriC ORC binds origins 2. Expose single-stranded templates (unwind) DnaA ORC? Mcm2-7? 3. Load helicase at nascent fork DnaC loads DnaB Cdc6, Cdt1 load Mcm2-7 Sld2, Sld3, Dpb11 load Cdc45 & GINS 4. Prime DNA synthesis Primase DNA Pola - primase Sld2, Sld3, Dpb11 loads DNA Pol  complex 5. Load polymerase(s) DnaB binds t subunit SSB & primer-template bind Clamp-Loader & Clamp ?? loads DNA Pol  complex

4. 2-Stage Model for Protein Assembly During Replication Initiation M Phase G1 Phase S Phase Cdc7-Dbf4 CDK Trigger License GINS Post-RC Pre-RC Pre-IC Initiation

5. Genetic Screens Enriching for Replication Initiation Mutants Conditional Mutants: cell division cycle (cdc) budded morphology 1N DNA content execution point before elongation

6. 1st shift HU 2nd shift ts 1st shift HU 2nd shift ts Initiation or Elongation?: Execution Point Analysis Requires independent and reversible means of inactivating two functions plus an “endpoint” assay A mutated initiation function is completed by the time elongation is blocked Initiation Elongation Cell Cycle Completed A mutated elongation function is still needed when elongation blocked Initiation Elongation Cell Cycle Remains Blocked HU = hydroxyurea which blocks replication elongation by inhibiting dNTPs biosynthesis

7. Genetic Screens Enriching for Replication Initiation Mutants Conditional Mutants: cell division cycle (cdc) budded morphology 1N DNA content execution point before elongation cdc6 cdc46/mcm5 cdc47/mcm7 cdc54/mcm4 cdc7 dbf4 cdc45

8. Genetic Screens Enriching for Replication Initiation Mutants Hypomorphic Mutants: minichromosome maintenance (mcm) Conditional Mutants: cell division cycle (cdc) % cells containing plasmid WITH selection % cells containing plasmid withOUT selection budded morphology 1N DNA content faster loss of minichromosome (I.e. selectable plasmid) from population suppression of mcm phenotype with multiple plasmid origins execution point before elongation cdc6 cdc46/mcm5 cdc47/mcm7 cdc54/mcm4 cdc7 dbf4 cdc45 cdc6 mcm2 mcm3 mcm5/cdc46 mcm10

9. SLD1 = DPB3 high copy suppression synthetic lethality SLD2 Loaders for Pol  Cdc45 & GINS DPB11 SLD3 SLD4 = CDC45 PSF1 SLD5 PSF2 coIP & mass spec PSF3 GINS complex CMG helicase holoenzyme subunits Gathering More Suspects: Guilt by Association genetic & physical interactions with replication genes/proteins Example: How Araki found many of the genes required for triggering initiation POL2 DPB2 DNA Pol  subunits

10. S. cerevisiae origin: ~120 bp ARS1 A B1 B2 B3 A is an essential ARS consensus sequence (ACS) B1, B2, & B3 partially redundant (linker scan)

11. Genetics: Hint from Mutational Correlation Poor in vitro binding activity ARS mutations Poor in vivo origin function Genetics: Establishing Initiation Function 2-D gel analysis of ARS1 initiation ORC5 orc5-1 Yeast Origin Recognition Complex (yORC): A 6 Subunit Initiator Biochem: Binding Activity ARS1 Footprint Note: most other eukaryotic ORCs do NOT have such sequence specificity

12. Genomic Footprint Chromatin IP (ChIP) Protein binding and/or distortion of specific sites Preferred binding sites of specific proteins Genomic Footprint DNA:yORC DNA yORC1ChIP preIP ARS1 control control ARS305 Gel control Microarray yORC1 ChIP-chip (chromosome VI) In Vivo Assays for Protein DNA Interactions Identifying intermediates in the assembly of initiation complexes on DNA

13. Pre-Replicative Complex (pre-RC) in G1 Phase Temporal analysis of genomic footprint at origins M G1 S-G2-M ORC hypersensitive site reduced in G1 phase Yeast 2µ origin Extended protection of B domainIn G1 phase Speculation: ORC binds origin throughout the cell cycle and is joined by other proteins in G1 phase to “license” origins for initiation

14. G1 S G2 M G1 Ordered Assembly of Proteins at Origins During G1 & S Using ChIP to establish temporal order and genetic dependencies of proteins assembling at the origin Example: G1-specific recruitment of Mcm7 is dependent on Cdc6 Synchronized yeast culture - Cdc6 or+ Cdc6 time points sampled for Mcm7 ChIP - Cdc6 + Cdc6 preIP S - G2 -M S - G2 -M G1 G1 control control ARS1 control

15. Using both Biochemistry and Genetics to understand function

16. Cdc45 ChIP-chip tracks with fork movement Dynamic Protein Associations Through G1 and S Combining temporal and spatial analysis of replication and binding in synchronized cells BrdU incorporation monitors fork movement Some replication proteins that load at origins later move with the forks: Mcm2-7, Cdc45, GINS, Mcm10, Dpb11, DNA Pol , DNA Pol , DNA Pol , PCNA (clamp), RFC1-5 (clamp loaders), RFA

17. 2-Stage Model for Protein Assembly During Replication Initiation M Phase G1 Phase S Phase Cdc7-Dbf4 CDK Trigger License GINS Post-RC Pre-RC Pre-IC Initiation

18. Helicase Activity Drosophila extract purify helicase activity Cdc45 - Mcm2-7 - GINS (CMG - helicase “holoenzyme”) Biochemical insights into Mcm loading and activation Pre-RC Assembly (helicase loading) Can control addition order of protein, cofactors, or inhibitors Can substitute mutant/modified proteins with altered activities Can analyze structures with greater resolution and accuracy Mcm2-7 doublehexamer remains on DNA after high salt wash ORC-DNA ATP Cdc6 Cdt1-Mcm2-7 EM reconstruction side end Replication Elongation Blocking Discussion Paper C N N C hexamer hexamer

19. 2-Stage Model for Protein Assembly During Replication Initiation M Phase G1 Phase S Phase Cdc7-Dbf4 CDK helicase holoenzyme loaded around unwound SS DNA core helicase loaded around DS DNA Trigger License GINS Post-RC Pre-RC Pre-IC Initiation

20. A Simplified View of Cyclin Dependent Kinases (CDKs) CDK regulation CDKs = kinase + cyclin cyclins undergo periodic synthesis and proteolysis different cyclins activate CDKs to promote different cell cycle events CDK in S. cerevisiae G1 cyclins not shown kinase = Cdc28 G1 cyclins = Cln1-3 S & M cyclins = Clb1-6 Molecular Biology of the Cell, 4th Ed.

21. Activation of CDKs and DDKs in S phase trigger origin initiation S G2 Dbf4-Cdc7 (DDK) Clb-Cdc28 (CDK) Pre-IC Post-RC Pre-RC Initiation

22. kinase substrate in vitro phosphorylated in vivo in kinase dependent manner in vitro and in vivo phosphorylation sites overlap Sld2 and Sld3 are essential replication targets for CDK triggering of initiation Sld2 and Sld3 phosphorylation promotes their independent binding to Dpb11 Sld2 phosphorylation promotes formation of “pre-Loading Complex” with GINS, pol , Dpb11 DDK Mcm4 and Mcm6 are essential targets for Cdc7-Dbf4 kinase (DDK) Identifying CDK and DDK targets for replication initiation How to identify in vivo targets of kinase? 1) Kinase substrate in vivo 2) (Necessity) Phosphorylated sites essential for kinase function 3) (Sufficiency??) Phosphomimic mutations allow bypass of kinase requirement

23. A temporal program regulates DNA replication within S phase Locate earliest DNA synthesis One example: Microarray analysis of copy number Earlier Initiation Later Initiation

24. Cdc45 Early Origins DDK CDK Late Origins Initiation Pre-RC Pre-RC Sld3 Temporal control of DNA replication through earlier DDK action? S G2 CDK DDK Post-RC Pre-RC (DDK activated) Initiation Post-RC What distinguishes earlier from later origins? What determines when a later origin becomes ready to fire? Why is there temporal control of DNA replication within S phase?

25. X X X X X X if you want a 50,000 origin genome to NOT re-initiate with 99.5% fidelity then re-initiation at each origin must be prevented with 99.99999% fidelity 50,000 (.9999999) = .995 Cell cycle control of origin function must be highly efficient CDK

26. preRC assembly NO preRC assembly CDK Sld2 Sld3 NO triggering initiation trigger initiation The CDK paradigm for once and only once replication

27. Sld2 Sld3 The CDK paradigm for once and only once replication preRC assembly Some preRC re-assembly CDK NO triggering initiation trigger initiation

28. CDKs Target Multiple Proteins to Block pre-RC Re-assembly Overlapping mechanisms ensure re-initiation is blocked at thousands of origins In budding yeast, CDK phosphorylation of 1) Mcm3 promotes Mcm2-7 nuclear exclusion 2) Cdc6 promote its proteolysis 3) Cdc6 promotes CDK binding and inhibition 4) Orc2/Orc6 inhibits recruitment of Cdt1-Mcm2-7 5) CDK binding to Orc6 inhibits ORC function The extensive overlap of mechanisms is conserved, NOT specific mechanisms Metazoans have additional CDK-independent mechanisms inhibiting re-initiation

29. X X X X X X if you want a 50,000 origin genome to NOT re-initiate with 99.5% fidelity then re-initiation at each origin must be prevented with 99.99999% fidelity 50,000 (.9999999) = .995 Cell cycle control of origin function must be highly efficient CDK

30. Aneuploidy Other Instability? Translocations? Inversions? Loss of Heterozgosity? How important is it to prevent re-initiation? Partial loss of replication control in yeast can greatly induce genomic instability Gene Amplification

31. Beyond Initiation: Keeping the Fork Going Through Thick and Thin Many genomic insults are now thought to originate from replication accidents DNA lesions induce responses to: (1) protect stalled forks (2) bypass lesions (3) delay further initiation (4) block cell cycle 2 3 1 4 Segurado & Tercero, Biol. Cell (2009) 11:617-627

32. Epigenetic Chromatin States -- how are chromatin states inherited during DNA replication? -- does replication timing contribute to this inheritance? Development and Differentiation -- do replication timing changes help execute developmental decisions? Sister Chromatid Cohesion -- how is the establishment of cohesion coupled to DNA replication? Meiotic Recombination -- how is initiation of DS breaks coupled to DNA replication? Cancer Biology -- does loss of replication control contribute to oncogenic genomic instability? Evolution -- does sporadic re-replication contribute to genetic variation? Expanding influence of replication on other processes

34. The Replication Checkpoint Insures Mitosis Does Not Proceed If Replication Is Delayed or Blocked Branched view of cell cycle Standard view of cell cycle termination elongation Replication initiation mec1 Mec1 rad53 Start Rad53 Segregation mitosis Passage through Start independently sets into motion both Replication and Segregation Replication is normally completed before mitosis can begin, allowing their proper temporal order Block or delay in elongation signals checkpoint mechanism (e.g. Mec1, Rad53) to prevent mitosis Exact nature of signal is unknown but it cannot be generated unless some initiation occurs (abnormal fork structure? stalled replisome?)

35. Checkpoint Mechanisms Also Stabilize Stalled Replication Forks Abnormal replication intermediates accumulate when replication is delayed in checkpoint mutants Unperturbed Replication dNTP Depleted Replication WT hemi-replicated large gaps collapsed forks Checkpoint Mutant (rad 53) Based on EM analysis by Sogo & Foiani Labs, Science (2002) 297:599 Possible functions of checkpoint mechanisms at forks during replicative “stress” maintain proper coordination of leading and lagging strand synthesis prevent fork collapse and branch migration These protective functions allow forks to resume replicating when the stress is removed

36. ~ 10 kb interorigin distance Origin Usage is Regulated During Development S Phase Length Developing Frog 20-25 min ~ 20 kb interorigin distance ~ 100 -200 kb interorigin distance ~ 6-8 hrs Line represents DS DNA

37. Origin Timing is Influenced by Chromatin Structure S. cerevisiae origins fire throughout S phase in a defined and reproducible order Telomeric heterochromatin can delay origin firing Position effect on origin timing ARS501 WT Sir dependent heterochromatin Chr 5 late TEL ARS1 Chr 4 early Y’ ARS fires late Swap positions sir mutant ARS1 Chr 5 late TEL Y’ ARS fires early ARS501 Chr 4 early from Gottschling Lab, Gene & Dev (1999) 13:146 from Brewer and Fangman labs, Cell (1992) 68:333

38. s Replication is Coupled to Establishment of Sister Chromatid Cohesion Speculative polymerase switch model Cohesin complexes (Scc1,Scc3,Smc1,Smc3) hold sisters together and ensure bipolar spindle attachment Cohesins must be present on chromatin during replication for proper cohesion to occur Replication-like proteins have been implicated in the establishment of cohesion: - two DNA polymerases of the s family, Trf4 and Trf5 - a modified RFC clamp loader with Ctf8, Ctf18, and Dcc1 substituting for Rfc1 figure from Hieter Lab, Mol Cell (2001) 7:959

39. Duplication of Nucleosome Structure During Eukaryotic Replication Parental nucleosomes distribute randomly to daughter DNA Model for new histone deposition at replication forks New histones complete the daughter complement of nucleosomes Histone Deposition Factors (CAF-1 and/or Asf1)are recruited to newly replicated DNA by PCNA and deposit H3H4 tetramers H2A-H2B dimers self assemble onto H3-H4 tetramers caf-1 asf1 double deletion mutants are viable in yeast, suggesting other histone deposition mechanisms exist How higher order chromatin structure is duplicated is not known

40. Does the replicator model apply to metazoan origin? The ARS plasmid assay has failed to identify metazoan origins Alternative strategy: physically map sites of initiation genetically identify sequences required for initiation at those sites example: potential origin in human globin gene cluster 100 kb E Gg Ag d b LCR IR Deletions in either region(from thallasemia patients) inactivates the IR IR = Initiation Region based on physical mapping of nascent strands LCR = Locus Control Region (required for transcriptional activity of entire cluster) Alternative hypothesis: little sequence specificity for initiation site zones of initiation established by chromatin structure

41. Eukaryotic Replication Initiation is Coupled to the Cell Cycle Helicase(?) Helicase(?) Loaders Primase Initiator 2 1 Kinases Polymerase Origin Recognition Complex pre-Replicative Complex (pre-RC) Replicative Complex (RC) Two fundamental stages of initiation defined primarily by Chromatin IP and Chromatin Association assays Stage 1: pre-RC assembly in G1 phase makes origins competent to initiate DNA replication Stage 2: Passage through Start (G1 commitment point) activates Clb-Cdc28 kinase and Cdc7-Dbf4 kinase which trigger formation of the replicative complex and initiation of DNA replication

42. Using both Biochemistry and Genetics to understand function

43. Structural Analysis of Intermediates Examples of structural features of intermediates that can be monitored Nucleic Acids Complexes Proteins Composition Cofactor (NTP) Status Size Conformation Stoichiometry Shape Modification Conformation DS versus SS Interacting Sequences Ligand Binding Strand Pairing Interacting Domains Covalent Linkages Modification Covalent Linkages Topology

44. 2-Stage Model for Protein Assembly During Replication Initiation M Phase G1 Phase S Phase CDK Cdc7-Dbf4 Trigger License GINS Post-RC Pre-RC Pre-IC Initiation

45. Using both Biochemistry and Genetics to understand function

46. Metazoans have CDK-independent mechanisms to block re-initiation

47. X X X X X X The cell cycle control of replication must be highly efficient Fidelity: > 99.999% block for genome with 50,000 origins Hayles et al. Cell 1994 Cyclin dependent kinases implicated