Biochemistry of Eukaryotic Replication Fork and DNA Repair

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Project Summary DNA replication is performed by numerous proteins that act as a dynamic machine, termed a replisome. The core components of the eukaryotic replisome consist of 1) an 11 subunit “CMG” helicase that separates the parental DNA strands, 2) The leading and lagging strand DNA polymerases (Pol), Pol d and Pol e, respectively,3) The PCNA sliding clamp that encircles DNA and tethers both Pols to DNA for high processivity, 4) the RFC clamp loader pentamer that loads PCNA onto DNA and 5) Pol a-primase that makes a hybrid RNA- DNA primed site for the Pols to initiate DNA synthesis. In addition to these “core” components, there are several ancillary proteins including RPA, Tof1, Mec1, Csm3, FACT, Mcm10, Ctf4, Ctf18-RFC. There are a host of proteins that assemble two CMGs around duplex DNA at origins. The CMG dimer unwinds the closed duplex in an unknown reaction and scaffolds enzymes to assemble replisomes. We have purified these proteins in the yeast (Saccharomyces cerevisiae; S.c.) system. In this proposal, we will extend our studies on the structure/function of the eukaryotic replisome. We will use biochemical and single-molecule methods to determine if PCNA accumulates on the lagging strand as expected, and whether PCNA may periodically be left on the leading strand for mismatch repair and assembly of naïve nucleosomes. We have solved numerous structures with our collaborator, Huilin Li (VanAndel Institute, MI), and have many more structures in progress and planned. We have purified the several factors of the ATR DNA damage checkpoint signaling system of which many replisome proteins are targets of this pathway. We plan biochemical studies that will clarify targets and their effect on replisomes. We will determine the mechanism of nucleosome inheritance during replication. In metazoans, epigenetic inheritance of nucleosomes, gone awry, can lead to cancer and other diseases. In yeast, cell studies have shown that a Mcm2 histone binding mutant prevents epigenetic transfer to lagging strands, and Pol e lacking the Dpb3/4 subunits does not transfer epigenetic marks to the leading strand. We plan to visualize nucleosome transfer during replication in real time using single-molecule studies with our newly acquired Q trap) in collaboration with Dr. Shixin Liu (Rockefeller University)). We have various nucleosome mobility factors and yeast nucleosomes having different fluorescently tagged histones for these studies. Replication occurs in nuclear foci, having “replication factories” with many DNA replication forks. Our recent biochemical and structural studies have defined the composition and atomic structure of the most basic unit of a replication factory, a dimeric replisome. We will employ super resolution microscopy to validate if our reconstituted factory is the same as that inside cells. We have insight into how duplex DNA at origins is opened into single strands from our recent finding that the twin CMG helicases encircling duplex DNA at an origin are directed inward, opposite the “outward” direction thought for a decade. We find that two inward directed CMG can shear DNA apart. We have plans to further this line of investigation.