Analyzing Origins and Regulation of Replication Initiation

1. Lecture 3: Origins and Initiation and Regulation Analyzing role and function of sequence elements Increasing the power of genetic tools with better in vivo molecular phenotypes Regulation through feedback inhibition by reaction products Regulation through cell cycle control inputs

2. Prokaryotic and Eukaryotic Replication Initiation Activities 5’ 3’ 5’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ Converting DS DNA to replication fork 1. Recognize initiation site (replication origin) 2. Expose single-stranded templates (unwind) 3. Load helicase at nascent fork 4. Prime DNA synthesis 5. Load polymerase(s)

3. Identifying Replicators (Genetic Mapping of Origins) ARS Assay Function: conferring autonomous maintenance on a plasmid

4. Bacteria have small well-defined origins S. cerevisiae origin: ~120 bpARS1 E. coli origin: 245 bporiC Chromatin Accessibility ORC Binding Initial Unwinding Initiator DnaA Loading A B1 B2 B3 9 9 13 13 13 9 9 9 l l l l l l l l l l l 9 DnaA 9-mer binding 13 A/T- rich 13-mer repeats GATC sites (for regulation) l A and B1: ORC binding B2 and B3: promote nucleosome free region?

5. Biochemical Dissection of OriC Initiation Develop in vitro system Establish “purified” system dnaA - Initiator: bind origin, unwind DNA, load helicase dnaB - helicase dnaC - deliver and loads helicase SSB - stabilizes unwound DNA dnaG - prime DNA synthesis gyrase - negatively supercoil DNA (facilitates unwinding) PolII holo - DNA synthesis Pol I, Rnase H, Ligase -process Okazaki fragments Create partial reactions and structurally analyze intermediates Infer protein function and develop specific assays

6. Model for oriC Initiation Bidirectional Replication

7. ATP is an allosteric regulator of DnaA oligomerization SS DNA binding unwinding

8. Prokaryotic and Eukaryotic Replication Initiation Activities 5’ 3’ 5’ 3’ 3’ 3’ 5’ 5’ 5’ 3’ Converting DS DNA to replication fork E. coli 1. Recognize initiation site (replication origin) DnaA binds oriC 2. Expose single-stranded templates (unwind) DnaA 3. Load helicase at nascent fork DnaC loads DnaB Primase 4. Prime DNA synthesis DnaB binds t subunit SSB & primer-template bind Clamp-Loader & Clamp 5. Load polymerase(s)

9. Multiple mechanisms inhibiting re-initiation of oriC 1)Origin Inactivation: Chromosomes are marked by dam methylation and become temporarily hemimethylated when they are replicated SeqA binding to hemimethylatedoriC blocks DnaA initiation function Me Me GATC dam GATC dam GATC CTAG CTAG CTAG Me replication

10. Exactly how seqA blocks oriC re-initiation is not known seqA binding to hemimethlyatedoriC somehow prevents dnaA initiation function without inhibiting dnaA high affinity binding to oriC

11. Multiple mechanisms inhibiting re-initiation of oriC 1)Origin Inactivation: Chromosomes are marked by dam methylation and become temporarily hemimethylated when they are replicated SeqA binding to hemimethylatedoriC blocks DnaAoligomerization Me Me GATC dam GATC dam GATC CTAG CTAG CTAG Me replication 2) Decreased Initiator Activity: DnaA-ATP is inactivated by ATP hydrolysis by -- Hda1 bound to a loaded sliding clamp -- binding to the DnaA binding element datA

12. Appealing model: nucleotide driven molecular switch DnaA-ATP is active initiator DnaA-ATP hydrolysis and inactivation is coupled to initiation DnaA-ATP is reset for next round of initiation Regulation of DnaA activity via nucleotide binding Binds oriC Unwinds oriC Load helicase + + + DnaA-ATP + - - DnaA-ADP + - - DnaA

13. Conversion of DnaA-ATP to DnaA-ADP In vivo evidence for the conversion Levels of ATP bound to DnaA cycle: high (~80%) just before initiation and low (~16%) soon after Biochemical purification of an ATPase stimulating activity HdaA stimulates DnaA ATPase activity when bound to a clamp loaded onto a primer-template junction This couples inactivation of DnaA to a late initiation event Genetic evidence for the relevance ATP hydrolysis Preventing ATP hydrolysis with a dnaA hydrolysis mutation or an hdaA deletion leads to -- accumulation of DnaA-ATP -- overreplication of DNA

14. DNA Synthesis The Challenge: coupling regulation of DnaA-ATP state to replication initiation cycles Clamp loading HdaA Regulatory Inactivation of DnaA RIDA DDAH datA-dependent DnaA-ATP Hydrolysis DARS DnaAReactivating Sequence IHF Integration Host Factor

15. 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) ORC binds origins DnaA binds oriC 2. Expose single-stranded templates (unwind) DnaA ORC? Mcm2-7? 3. Load helicase at nascent fork DnaC loads DnaB Cdc6 & Cdt1 load Mcm2-7 4. Prime DNA synthesis Primase DNA Pola - primase Mcm10? Cdc45-Sld3 Dpb11-Sld2 GINS complex DnaB binds t subunit SSB & primer-template bind Clamp-Loader & Clamp 5. Load polymerase(s)

16. Budding yeast also have small well-defined origins S. cerevisiae origin: ~120 bpARS1 E. coli origin: 245 bporiC Chromatin Accessibility ORC Binding Initial Unwinding Initiator DnaA Loading A B1 B2 B3 9 9 13 13 13 9 9 9 l l l l l l l l l l l 9 DnaA 9-mer binding 13 A/T- rich 13-mer repeats GATC sites (for regulation) l A and B1: ORC binding B2 and B3: promote nucleosome free region?

17. Yeast origins have a nucleosomal structure Nucleosome positions relative to ORC binding sites aligned for 219 origins White – nucleosome occupied Black – nucleosome free

18. Eukaryotic origins appear “redundant” Multiple deletions have little overall effect on chromosome replication and cell division But increase the probability of rare rearrangements ARSs X X X Origin Use By 2-D Gel S.cerevisiae Chromosome 3 Origins

19. Identifying Sites of Initiation (Physical Mapping of Origins) Example: Map the earliest DNA synthesis in a region

20. Chromatin Structure nucleosome free region Higher eukaryotic origins may be defined by chromatin Sequence Recognition In Yeast ORC binds ACS ORC binds nonspecifically to AT rich sequence chromatin may be primary origin determinant In Metazoans

21. Bioregulation through regulated protein assembly M Phase G1 Phase S Phase Cdc7-Dbf4 CDK Trigger License GINS Post-RC Pre-RC Pre-IC 2-stage model for eukaryotic replication initiation Initiation

22. 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

23. 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

24. 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 is blocked Initiation Elongation Cell Cycle Remains Blocked HU = hydroxyurea which blocks replication elongation by inhibiting dNTPs biosynthesis

25. Biochem: Binding Activity ARS1 Footprint Note: most other eukaryotic ORCs do NOT have such sequence specificity A Yeast Initiator Protein: Guilt by Association S. cerevisiae origin: ~120 bp ARS1 Help ORC bind on chromatin ORC Binding on naked DNA A B1 B2 B3 A is an essential ARS consensus sequence

26. 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

27. 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

28. 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

29. 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 Cell Cycle Time 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

30. 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

31. Helicase Activity Drosophila extract purify helicase activity Cdc45 - Mcm2-7 - GINS (CMG - helicase “holoenzyme”) Biochemical insights into Mcm loading and activation Pre-RC Assembly Assay (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 C N N C hexamer hexamer

32. 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

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

34. Cdc45 Early Origins DDK CDK Late Origins Initiation Pre-RC Pre-RC Sld3 Temporal control of DNA replication through earlier DDK action? Cdc7-Dbf4 Kinase 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?

35. 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

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

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

38. 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

39. 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