Reconstitution Of Regulated Eukaryotic Dna Replication With Purified Budding Yeast Proteins

DNA replication is a fundamental process by which organisms copy their DNA. Biochemical reconstitution approaches in bacteria and viral systems have been instrumental in enhancing our mechanistic understanding of this process. However, a reconstituted system to study eukaryotic chromosomal DNA replication has been lacking. My thesis work focused on overcoming this deficiency by aiming to reconstitute eukaryotic DNA replication with purified budding yeast proteins, as chromosome replication is highly conserved from yeast to humans but best understood in budding yeast, Saccharomyces cerevisiae. Eukaryotic DNA replication initiates via a conserved two-step mechanism from multiple origin sites distributed along the length of each chromosome. In the first step, which is restricted to G1 phase, the core of the replicative helicase complex, Mcm2-7, is loaded in an inactive form at origins of replication, forming a pre-replicative complex (pre-RC). Subsequent activation of the Mcm2-7 helicase in the second step occurs exclusively in S-phase. Mcm2-7 loading has been previously reconstituted with purified proteins. However, the unusual double hexameric configuration of the Mcm2-7 complex bound around double stranded DNA observed in this reaction, while providing a molecular rationale for bi-directional origin activation, raised the question whether this structure is a true replication intermediate. Here, I show that reconstituted pre-RCs indeed support regulated replication of plasmid DNA in yeast cell extracts exhibiting the hallmarks of cellular replication initiation. Expanding on this observation, I have subsequently reconstituted DNA replication free in solution with purified budding yeast proteins in order to generate a system that allows for the biochemical study of all steps of eukaryotic DNA replication. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ and Pol ε and canonical maturation of Okazaki fragments into continuous daughter strands. A dual regulatory role for chromatin was uncovered during the replication of chromatinized templates: i) promoting origin dependence and ii) determining Okazaki fragment length by restricting Pol δ progression. Hence, this system provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication

Rad53 controls DNA unwinding after helicase-polymerase uncoupling at DNA replication forks

ABSTRACTThe coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Using the reconstituted budding yeast DNA replication system, we demonstrate that Pol ε variants harboring catalytic point mutations in the Pol2 polymerase domain, contrary to Pol2 polymerase domain deletions, inhibit DNA synthesis at replication forks by displacing Pol δ from PCNA/primer-template junctions, causing excessive DNA unwinding by the replicative DNA helicase, CMG, uncoupled from DNA synthesis. Mutations that suppress the inhibition of Pol δ by Pol ε restore viability in Pol2 polymerase point mutant cells. We also observe uninterrupted DNA unwinding at replication forks upon dNTP depletion or chemical inhibition of DNA polymerases, demonstrating that leading strand synthesis is not tightly coupled to DNA unwinding by CMG. Importantly, the Rad53 kinase controls excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork-stabilization by the replication checkpoint.</jats:p

Multistep loading of PCNA onto DNA by RFC

AbstractThe DNA sliding clamp proliferating cell nuclear antigen (PCNA) is an essential co-factor for many eukaryotic DNA metabolic enzymes. PCNA is loaded around DNA by the ATP-dependent clamp loader replication factor C (RFC), which acts at single-stranded/double-stranded DNA junctions harboring a recessed 3’ end (3’ ss/dsDNA junctions) and at DNA nicks. To illuminate the loading mechanism we have investigated the structure of RFC:PCNA bound to ATPγS and 3’ ss/dsDNA junctions or nicked DNA using cryogenic electron microscopy. Unexpectedly, we observe open and closed PCNA conformations in the RFC:PCNA:DNA complex, revealing that PCNA can adopt an open, planar conformation that allows direct insertion of dsDNA, and indicating that PCNA ring-closure is not mechanistically coupled to ATP-hydrolysis. By resolving multiple DNA-bound states of RFC:PCNA we observe that partial melting facilitates lateral insertion into the central channel formed by RFC:PCNA. We also resolve the Rfc1 N-terminal domain and demonstrate that its single BRCT domain participates in coordinating DNA prior to insertion into the central RFC channel, which promotes PCNA loading on the lagging strand of replication forks in vitro. Combined, our data suggest a comprehensive and fundamentally revised model for the RFC-catalyzed loading of PCNA onto DNA.</jats:p

A hypomorphic mutation in <i>Pold1</i> disrupts the coordination of embryo size expansion and morphogenesis during gastrulation

AbstractFormation of a properly sized and patterned embryo during gastrulation requires a well-coordinated interplay between cell proliferation, lineage specification and tissue morphogenesis. Following transient physical or pharmacological manipulations, pre-gastrulation stage mouse embryos show remarkable plasticity to recover and resume normal development. However, it remains unclear how mechanisms driving lineage specification and morphogenesis respond to defects in cell proliferation during and after gastrulation. Null mutations in DNA replication or cell-cycle related genes frequently lead to cell cycle arrest and reduced cell proliferation, resulting in developmental arrest before the onset of gastrulation; such early lethality precludes studies aiming to determine the impact of cell proliferation on lineage specification and morphogenesis during gastrulation. From an unbiased ENU mutagenesis screen, we discovered a mouse mutant, tiny siren (tyrn), that carries a hypomorphic mutation producing an aspartate to tyrosine (D939Y) substitution in Pold1, the catalytic subunit of DNA polymerase δ. Impaired cell proliferation in the tyrn mutant leaves anterior-posterior patterning unperturbed during gastrulation but results in an overall reduction in embryo size and in severe morphogenetic defects. Our analyses show that the successful execution of morphogenetic events during gastrulation requires that lineage specification and the ordered production of differentiated cell types occur in concordance with embryonic growth.Summary statementPold1 hypomorphic mutation caused reduced size and abnormal morphology of gastrulating mouse embryos, supporting the importance of coordinated embryo size, lineage specification and tissue morphogenesis for normal embryogenesis.</jats:sec

DNA polymerase ε relies on a unique domain for efficient replisome assembly and strand synthesis

AbstractDNA polymerase epsilon (Pol ε) is required for genome duplication and tumor suppression. It supports both replisome assembly and leading strand synthesis; however, the underlying mechanisms remain to be elucidated. Here we report that a conserved domain within the Pol ε catalytic core influences both of these replication steps in budding yeast. Modeling cancer-associated mutations in this domain reveals its unexpected effect on incorporating Pol ε into the four-member pre-loading complex during replisome assembly. In addition, genetic and biochemical data suggest that the examined domain supports Pol ε catalytic activity and symmetric movement of replication forks. Contrary to previously characterized Pol ε cancer variants, the examined mutants cause genome hyper-rearrangement rather than hyper-mutation. Our work thus suggests a role of the Pol ε catalytic core in replisome formation, a reliance of Pol ε strand synthesis on a unique domain, and a potential tumor-suppressive effect of Pol ε in curbing genome re-arrangements.</jats:p

Checkpoint Kinase Rad53 Couples Leading- and Lagging-Strand DNA Synthesis under Replication Stress

The checkpoint kinase Rad53 is activated during replication stress to prevent fork collapse, an essential but poorly understood process. Here we show that Rad53 couples leading- and lagging-strand synthesis under replication stress. In rad53-1 cells stressed by dNTP depletion, the replicative DNA helicase, MCM, and the leading-strand DNA polymerase, Pol ε, move beyond the site of DNA synthesis, likely unwinding template DNA. Remarkably, DNA synthesis progresses further along the lagging strand than the leading strand, resulting in the exposure of long stretches of single-stranded leading-strand template. The asymmetric DNA synthesis in rad53-1 cells is suppressed by elevated levels of dNTPs in vivo, and the activity of Pol ε is compromised more than lagging-strand polymerase Pol δ at low dNTP concentrations in vitro. Therefore, we propose that Rad53 prevents the generation of excessive ssDNA under replication stress by coordinating DNA unwinding with synthesis of both strands.</p