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

2. Prokaryotic and Eukaryotic Replication Initiation Activities 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) 5’5’ 5’5’ 3’3’ 3’3’ 5’5’ 3’3’ 5’5’ 5’5’ 3’3’ 3’3’ Converting DS DNA to replication fork

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

4. Bacteria have small well-defined origins E. coli origin: 245 bp oriC S. cerevisiae origin: ~120 bp ARS1 13 A/T- rich 13-mer repeats Initiator DnaA Loading 9 99 9 DnaA 9-mer binding Initial Unwinding AB1B2B3 ORC Binding A and B1: ORC binding 9 13 lllllllllll l GATC sites (for regulation) Chromatin Accessibility B2 and B3: promote nucleosome free region? 9

5. Create partial reactions and structurally analyze intermediates Biochemical Dissection of OriC Initiation 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 Develop in vitro system Establish “purified” system 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 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) 5’5’ 5’5’ 3’3’ 3’3’ 5’5’ 3’3’ 5’5’ 5’5’ 3’3’ 3’3’ DnaA binds oriC DnaA DnaC loads DnaB Primase E. coliConverting DS DNA to replication fork DnaB binds t subunit SSB & primer-template bind Clamp-Loader & Clamp

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

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

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

12. Binds oriC Regulation of DnaA activity via nucleotide binding Unwinds oriCLoad helicase DnaA-ATP ++ + DnaA-ADP +-- DnaA +-- 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

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

14. The Challenge: coupling regulation of DnaA-ATP state to replication initiation cycles Clamp loading RIDA Regulatory Inactivation of DnaA DNA Synthesis DDAHdatA-dependent DnaA-ATP Hydrolysis DARSDnaA Reactivating Sequence IHFIntegration Host Factor HdaA

15. Prokaryotic and Eukaryotic Replication Initiation Activities 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) 5’5’ 5’5’ 3’3’ 3’3’ 5’5’ 3’3’ 5’5’ 5’5’ 3’3’ 3’3’ DnaA binds oriC ORC binds origins DnaA DnaC loads DnaBCdc6 & Cdt1 load Mcm2-7 Primase DNA Pol  - primase E. coliS. cerevisiae ORC? Mcm2-7? Mcm10? Cdc45-Sld3 Dpb11-Sld2 GINS complex Converting DS DNA to replication fork DnaB binds  subunit SSB & primer-template bind Clamp-Loader & Clamp

16. Budding yeast also have small well-defined origins E. coli origin: 245 bp oriC S. cerevisiae origin: ~120 bp ARS1 13 A/T- rich 13-mer repeats Initiator DnaA Loading 9 99 9 DnaA 9-mer binding Initial Unwinding AB1B2B3 ORC Binding A and B1: ORC binding 9 13 lllllllllll l GATC sites (for regulation) Chromatin Accessibility B2 and B3: promote nucleosome free region? 9

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

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

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

21. Bioregulation through regulated protein assembly Pre-RCPre-ICPost-RC Initiation CDKCdc7-Dbf4 License Trigger GINS M PhaseG1 PhaseS Phase 2-stage model for eukaryotic replication initiation

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

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

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

25. A Yeast Initiator Protein: Guilt by Association S. cerevisiae origin: ~120 bp ARS1 AB1B2B3 ORC Binding on naked DNA A is an essential ARS consensus sequence Help ORC bind on chromatin Biochem: Binding Activity ARS1 Footprint Note: most other eukaryotic ORCs do NOT have such sequence specificity

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

27. Pre-Replicative Complex (pre-RC) in G1 Phase Temporal analysis of genomic footprint at origins MG1S-G2-M ORC hypersensitive site reduced in G1 phase Extended protection of B domainIn G1 phase Yeast 2µ origin Speculation: ORC binds origin throughout the cell cycle and is joined by other proteins in G1 phase to “license” origins for initiation

28. Ordered Assembly of Proteins at Origins During G1 & S Using ChIP to establish temporal order and genetic dependencies of proteins assembling at the origin ARS1 control G1 S - G2 -M G1 S - G2 -M - Cdc6+ Cdc6 preIP Example: G1-specific recruitment of Mcm7 is dependent on Cdc6 time points sampled for Mcm7 ChIP G1MG2SG1 Synchronized yeast culture - Cdc6 or + Cdc6

29. Dynamic Protein Associations Through G1 and S Combining temporal and spatial analysis of replication and binding in synchronized cells 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 BrdU incorporation monitors fork movementCdc45 ChIP-chip tracks with fork movement Cell Cycle Time

30. 2-Stage Model for Protein Assembly During Replication Initiation Pre-RCPre-ICPost-RC Initiation License Trigger GINS M PhaseG1 PhaseS Phase CDKCdc7-Dbf4

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

32. 2-Stage Model for Protein Assembly During Replication Initiation Pre-RCPre-ICPost-RC Initiation License Trigger GINS M PhaseG1 PhaseS Phase CDKCdc7-Dbf4 core helicase loaded around DS DNA helicase holoenzyme loaded around unwound SS DNA

33. Activation of CDKs and DDKs in S phase trigger origin initiation Clb-Cdc28 (CDK) Dbf4-Cdc7 (DDK) Pre-RCPost-RC Pre-IC Initiation G2S Cdc7-Dbf4 Kinase

34. Temporal control of DNA replication through earlier DDK action? CDK DDK Pre-RC (DDK activated) Post-RC Initiation G2S Post-RC Pre-RC DDKCDK Pre-RC Initiation Early Origins Late Origins 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? Cdc45 Sld3 Cdc7-Dbf4 Kinase

35. Cell cycle control of origin function must be highly efficient XXX XXX 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 (.9999999) 50,000 =.995 CDK

36. preRC assembly NO triggering initiation NO preRC assembly trigger initiation CDK Sld2Sld3 The CDK paradigm for once and only once replication

37. preRC assembly NO triggering initiation Some preRC re-assembly trigger initiation CDK Sld2Sld3 The CDK paradigm for once and only once replication

38. CDKs Target Multiple Proteins to Block pre-RC Re-assembly In budding yeast, CDK phosphorylation of 1) Mcm3 promotes Mcm2-7 nuclear exclusion 2) Cdc6 promote its proteolysis 4) Orc2/Orc6 inhibits recruitment of Cdt1-Mcm2-7 Overlapping mechanisms ensure re-initiation is blocked at thousands of origins 3) Cdc6 promotes CDK binding and inhibition 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. Gene Amplification Partial loss of replication control in yeast can greatly induce genomic instability How important is it to prevent re-initiation? AneuploidyOther Instability? Translocations? Inversions? Loss of Heterozgosity?