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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 ordered assembly of protein machines Studying the inhibition/activation of activities to uncover regulatory mechanisms Activities needed to initiate DNA replication 5’ 3’ 3’ 5’ 5’ helicase DNA polymerase 3’ Converting DS DNA to a replication fork Activities needed to initiate DNA replication 5’ origin 3’ 5’ 3’ helicase Converting DS DNA to a replication fork 1. Recognize initiation site (replication origin) 2. Expose single-stranded templates (unwind) 3. Load helicase at nascent fork 4. Prime DNA synthesis Activities needed to initiate DNA replication 5’ origin 3’ 5’ 3’ helicase DNA polymerase Converting DS DNA to a 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 DNA polymerases and start DNA synthesis Identifying origins genetically (replicator elements) ARS Assay Function: conferring autonomous maintenance on a plasmid Using both Biochemistry and Genetics to understand function Sequence Element recover from cells protein-DNA binding introduce into cells Sequence Element in vitro mutagenesis Bacteria and Yeast have small well-defined origins E. coli origin: 245 bp oriC Initial Unwinding l l l l ORC Binding Initiator DnaA Loading 9 13 13 13 9 l l l 9 l l l S. cerevisiae origin: ~120 bp ARS1 9 9 A B1 Chromatin Accessibility B2 B3 l 9 DnaA 9-mer high affinity binding 13 A/T- rich 13-mer repeats l GATC sites (for regulation) A and B1: ORC binding B2 and B3: promote nucleosome free region? Biochemical Dissection of OriC Initiation Develop in vitro system Establish “purified” system DnaA DnaB DnaC DnaG Pol III holo SSBP Gyrase Pol I, Rnase H, Ligase Create partial reactions - structurally analyze intermediates - determine protein and cofactor dependencies Infer protein function and develop specific assays Simplified Model for oriC Initiation DnaA - binds origin, unwinds DNA, loads helicase DnaB - helicase DnaC - delivers and loads helicase DnaG - primes DNA synthesis Pol III holo - polymerase and clamp (polymerase) loading SSBP - stabilizes SS DNA Gyrase - negatively supercoil DNA (facilitates unwinding) Pol I, Rnase H, Ligase - process Okazaki fragments Bidirectional Replication ATPases Associated with a variety of cellular Activities (AAA+ proteins) With ATP bound in subunit interfaces nucleotide state can be coupled to large conformational change Many replication proteins form AAA+ ATPase Complexes Arginine promotes ATP hydrolysis ATP Pocket origin binding DnaA ORC helicase loading DnaC Cdc6 helicase DnaB Mcm2-7 clamp loader g complex RFC1-5 Schematic of Mcm2-7 hexamer organization Oligomerization of DnaA-ATP bound to oriC promotes unwinding high affinity binding low affinity binding oligomerization SS DNA binding Activities needed to initiate DNA replication 5’ origin 3’ 5’ 3’ helicase DNA polymerase Converting DS DNA to a replication fork E. coli 1. Recognize initiation site (replication origin) DnaA (oriC) 2. Expose single-stranded templates (unwind) DnaA 3. Load helicase at nascent fork DnaC, DnaA > DnaB 4. Prime DNA synthesis Primase 5. Load DNA polymerases Pol III Holoenzyme Coordinating replication initiation with E. coli cell cycle oriC terminus replication initiation Initiation occurs once per cell cycle at fixed ratio of cell volume per chromosome - Wallden et al. 2016 Cell 166:729-739 Multiple mechanisms inhibit re-initiation of oriC 1) Origin Inactivation: Chromosomes are marked by dam methylation and become temporarily hemimethylated when they are replicated. SeqA binding to hemimethylated oriC blocks DnaA low affinity binding required for initiation Me GATC CTAG dam GATC CTAG Me dam GATC CTAG Me replication SeqA binding to hemimethylated GATC prevents oriC re-initiation oriC replication immediately leads to hemimethlyation of its GATC sites SeqA binds specifically to hemimethlyated GATC, preventing DnaA-ATP binding to low affinity binding sites in oriC and thereby blocking immediate re-initiation SeqA binding also transiently protects hemimethylated GATC in oriC from dam methylase, but this protection only lasts for 1/3 of the cell cycle, after which full methylation and SeqA release occurs Coordinating replication initiation with E. coli cell cycle oriC terminus replication initiation SeqA inhibition by oriC inactivation 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 hemimethylated oriC blocks DnaA low affinity binding required for initiation Me GATC CTAG dam GATC CTAG Me dam GATC CTAG Me replication 2) Decreased Initiator Activity: DnaA-ATP is inactivated by ATP hydrolysis. It’s slow intrinsic hydrolytic activity is greatly stimulated by -- Hda1 bound to a loaded sliding clamp -- binding to the datA sequence element in the presence of IHF protein Regulation of DnaA activity via nucleotide binding Binds oriC Binds oriC High Affinity Sites Low Affinity Sites Unwinds oriC Load helicase DnaA-ATP + + + + DnaA-ADP + - - - DnaA + - - - Appealing model: nucleotide driven molecular switch DnaA-ATP is active initiator; DnaA-ADP and DnaA are inactive DnaA-ATP hydrolysis and inactivation is coupled to initiation DnaA-ADP release allows ATP rebind for next round of initiation Hda: initiation induced activity that converts DnaA-ATP to DnaA-ADP Initiation Coordinating replication initiation with E. coli cell cycle How is DnaA reactivated? oriC terminus How is it coupled to volume/chromosome ratio? replication initiation Hda and datA inhibition by DnaA inactivation SeqA inhibition by oriC inactivation Activities needed to initiate DNA replication 5’ origin 3’ 5’ 3’ helicase DNA polymerase Converting DS DNA to a replication fork S. cerevisiae 1. Recognize initiation site (replication origin) ORC 2. Expose single-stranded templates (unwind) ??? (Cdc45 - Mcm2-7 - GINS?) 3. Load helicase at nascent fork ORC,Cdc6,Cdt1 > Mcm2-7 Sld2,Sld3,Dpb11 > Cdc45, GINS 4. Prime DNA synthesis Pola-Primase 5. Load DNA polymerases Sld2,Sld3,Dpb11 > Pole ??? > Pold Bacteria and Yeast have small well-defined origins E. coli origin: 245 bp oriC Initial Unwinding l l l l ORC Binding Initiator DnaA Loading 9 13 13 13 9 l l l 9 l l l S. cerevisiae origin: ~120 bp ARS1 9 9 A B1 Chromatin Accessibility B2 B3 l 9 DnaA 9-mer binding 13 A/T- rich 13-mer repeats l GATC sites (for regulation) A and B1: ORC binding B2 and B3: promote nucleosome free region? Identifying origins physically (mapping initiation sites) 1) Map the sites of earliest DNA synthesis in a region 2) Map sites where replication bubbles first form Eukaryotic origins: lessons from yeast - chromosomal context affects origin activity and timing only some ARSs on plasmids are efficient origins in chromosomes - multiple origins are spaced closely enough to back up each other - but they are not redundant for long-term chromosome propagation Multiple deletions do not grossly affect chromosome replication and cell division But increase the probability of rare rearrangements ARSs X X Origin Use By 2-D Gel X 1,000 kb 2,000 kb S.cerevisiae Chromosome 3 Origins 3,000 kb Metazoan origins may be defined by chromatin more than sequence Sequence Recognition In Yeast In Metazoans Chromatin Structure ORC binds ACS nucleosome free region ORC binds nonspecifically to AT rich sequences chromatin accessibility may be primary origin determinant Better in vivo assays allow genetic analysis of eukaryotic replication E. Coli oriC S. cerevisiae ARS Develop in vitro system Genetically identify initiation factors Establish “purified” system Localize factors to origins and/or replication forks Create partial reactions and structurally analyze intermediates Establish order of assembly during initiation and cell cycle progression Infer protein function and develop specific assays Develop in vitro system and specific assays Future mechanistic studies (great Bioreg proposals) 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 yORC1 ChIP preIP control control Gel ARS305 Microarray or Sequencing control yORC1 ChIP-chip (chromosome VI) 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 G1 S G2 M G1 time points sampled for Mcm7 ChIP - Cdc6 G1 S - G2 -M + Cdc6 G1 S - G2 -M preIP control control ARS1 control Dynamic Protein Associations Through G1 and S Combining temporal and spatial analysis of replication and binding in synchronized cells Cdc45 ChIP-chip tracks with fork movement Cell Cycle Time 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 a, DNA Pol d, DNA Pol e, PCNA (clamp), RFC1-5 (clamp loaders), RFA 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 Biochemical analysis of Mcm loading and activation Pre-RC Assembly (helicase loading) Mcm2-7 doublehexamer remains on DNA after high salt wash ORC-DNA ATP Helicase Activity Drosophila extract purify helicase activity Cdc6 Cdt1-Mcm2-7 Cdc45 - Mcm2-7 - GINS (CMG - helicase “holoenzyme”) EM reconstruction side C N hexamer N end C hexamer 2-Stage Model for Protein Assembly During Replication Initiation M Phase G1 Phase S Phase Cdc7-Dbf4 License core helicase loaded around DS DNA CDK Trigger helicase holoenzyme loaded around unwound SS DNA GINS Post-RC Pre-RC Pre-IC Initiation Cell Cycle Inputs: activation of CDKs and DDKs trigger origin initiation Cdc7-Dbf4 Kinase S Dbf4-Cdc7 (DDK) Post-RC Pre-RC Clb-Cdc28 (CDK) Pre-IC Initiation G2 Identifying CDK and DDK targets for replication initiation How to show a protein is a biologically relevant target of a kinase? kinase substrate in vitro 1) Kinase substrate in vivo phosphorylated in vivo in kinase dependent manner in vitro and in vivo phosphorylation sites overlap 2) (Necessity) Phosphorylated sites essential for kinase function 3) (Sufficiency??) Phosphomimic mutations allow bypass of kinase requirement 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 e, Dpb11 DDK Mcm4 and Mcm6 are essential targets for Cdc7-Dbf4 kinase (DDK) Regulation of eukaryotic replication initiation Dependence on completion of G1 phase dependence on Cdc7 and CDK kinases Timing of origin firing within S phase see slide in appendix Preventing re-initiation within a single cell cycle example: origins/cell = 50,000 fidelity per origin = 0.9999999 fidelity per cell = (0.9999999) 50,000 = 0.995 Many mechanisms inhibit re-licensing and prevent re-initiation origin activation inhibit licensing inhibit licensing origin licensing inhibit licensing DDB1-Cul4 PCNA GEMININ Replication control is critical for genome stability Gene Amplification Aneuploidy Other Instability? Translocations? Inversions? Loss of Heterozgosity? DNA replication is coordinated with other genomic processes Replication of chromatin and chromatin states -- nucleosome structure and chromatin states are disrupted by the replication fork and must be faithfully duplicated after passage of the replication fork -- changes in chromatin states are associated with changes in replication timing Transcription -- prokaryotic replisomes can survive collisions with the transcription machinery -- but genomes still appear to avoid head on collisions between replication and transcription from strongly expressed genes. DNA Damage Repair -- Replication provides genome surveillance Sister Chromatid Cohesion -- establishment of cohesion is coupled to DNA replication Meiotic Recombination -- the DS breaks that initiate meiotic recombination are coupled to DNA replication Bioreg 2017 Lecture 3 Replication APPENDIX Yeast origins maintain a nucleosome free region Nucleosome positions relative to ORC binding sites aligned for 219 origins White – nucleosome occupied Black – nucleosome free Genetic Screens Enriching for Replication Initiation Mutants Conditional Mutants: cell division cycle (cdc) Hypomorphic Mutants: minichromosome maintenance (mcm) % cells containing plasmid WITH selection % cells containing plasmid withOUT selection budded morphology 1N DNA content faster loss of selectable plasmid because of higher chance for plasmid replication failure execution point before elongation suppression of mcm phenotype with multiple plasmid origins cdc6 cdc46/mcm5 cdc47/mcm7 cdc54/mcm4 cdc7 dbf4 cdc45 cdc6 mcm2 mcm3 mcm5/cdc46 mcm10 Initiation or Elongation?: Execution Point Analysis by Reciprocal Shift Requires independent and reversible means of inactivating two functions plus an “endpoint” assay Initiation: mutated gene has executed its function by the time DNA synthesis occurs DNA synthesis 1st shift ts Experiment 1 2nd shift HU 1st shift HU Experiment 2 2nd shift ts Elongation: mutated gene is still needed to function during DNA synthesis DNA synthesis 1st shift ts Experiment 1 2nd shift HU 1st shift HU Experiment 2 2nd shift ts Origin initiation timing within S phase is regulated Earlier Origins Earlier Replication Later Origins This timing correlate with transcriptional program and cell fate Embryonic Stem Cells Neural Precursor Cells The CDK paradigm for once and only once replication preRC assembly NO preRC assembly CDK NO triggering initiation Sld2 Sld3 trigger initiation The CDK paradigm for once and only once replication preRC assembly Some preRC re-assembly CDK NO triggering initiation Sld2 Sld3 trigger initiation You have a 100 bp sequence element whose function you can quantitatively assay after introducing it into cells You have a protein that you purified based on its ability to bind the sequence element and you wonder whether this protein is required for the function of the sequence element. Hence, you plan on identifying the gene that encodes the protein and mutagenizing the gene in cells where you can functionally assay the sequence element However, before you take all the effort to clone the gene and do the reverse genetics, you want some indication that this binding protein is probably important for the function of the sequence element. What quicker experiment can you do using available assays and reagents to increase your confidence that your protein is functionally relevan