Duplication of the genetic matierial is performed by a multiprotein replisome machine. The replisome of E. coli, the only cellular replisome that can be reconstituted in vitro, contains DnaB helicase, a homohexamer that unwinds DNA, primase, to initiate lagging strand fragments, and DNA polymerase III (Pol III) holoenzyme that utilizes two polymerases for concurent leading and lagging strand synthesis. The holoenzyme is composed of three major components: 1) 2-3 catalyitic Pol III cores, 2) b clamps that encircle DNA for high processivity, and 3) a clamp loader that places b clamps on primed sites. The replisome is organized by t subunits of the clamp loader that bind Pol III core and DnaB. During replication of long chromosomes the replisome encounters a variety of obsticals. The replisome can not fail in passing obsticals, unlike machines that make RNA or protein where intermittent failure is not a matter of life and death. One replisome obstical is RNA polymerase, one of the tightest DNA binding proteins. We examine the detailed mechanism by which the replisome displaces a codirectional RNA polymerase and how it recruits the mRNA for synthesis. We also propose the study of head- on collisions with arrays of RNA polymerase, and to identify helicases that help the replisome resolve these collisions. Another obstical is a DNA lesion, which will halt the high fidelity Pol III. We find that translesion polymerases, Pols II and IV, take over the replisome and slow the helicase to give time for DNA repair and move the replisome past blocks. We will apply single-molecule microscopy, along with ensemble assays, to understand the mechanism of this important process. Recent studies show the holoenzyme contains 3 Pol III cores. The 3rd Pol III core may be used to bypass lesions, which we will test. Because the clamp loader is an asymmetric structure it confers asymmetry onto the Pol III cores. We will also use single-molecule methods to determine which Pol III cores function on leading and lagging strands. During lagging strand synthesis, Pol III core is sometimes signaled to release from DNA before an Okazaki fragment is complete, leaving a ssDNA gap. We will study the source of the signal, size and frequency of ssDNA gaps, and whether the 3rd Pol III core participates in this post-replication repair. Chromosome duplication is a central life process, and therefore the replisome offers an ideal target for antibacterial compounds. We have identified and solved the structure of a small compound inhibitor bound to the b clamp and propose to expand this project, performing chemistry in collaboration with the Broad Institute. We also propose to use Rockefeller University's high throughput screening facility to identify inhibitors to other replisome components. Cellular replisomes from all three domains of life share similar strategies to the E. coli replisome. Hence, the lessons learned from this proposal should generalize. Finally, replication gone awry forms the basis for diseased states, including cancer. Understanding the basic processes of chromosome duplication may aid development of drugs for therapy of diseased states that involve the replication process.

Public Health Relevance

Chromosome duplication is a crucial life process in all cells and is performed by a mulitprotein replisome machine. This proposal explores the mechanism of the bacterial replisome in molecular detail. The essential nature of the replisome makes it an attractive target for antibacterial compounds and understanding the bacterial replisome should teach us the fundamental mechanism used to replicate human cells which may provide insight into cancer and treatment of diseases that involve abarrent replication.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Reddy, Michael K
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Rockefeller University
Other Domestic Higher Education
New York
United States
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Langston, Lance; O'Donnell, Mike (2017) Action of CMG with strand-specific DNA blocks supports an internal unwinding mode for the eukaryotic replicative helicase. Elife 6:
Georgescu, Roxana; Langston, Lance; O'Donnell, Mike (2015) A proposal: Evolution of PCNA's role as a marker of newly replicated DNA. DNA Repair (Amst) 29:4-15
Georgescu, Roxana E; Schauer, Grant D; Yao, Nina Y et al. (2015) Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation. Elife 4:e04988
Sun, Jingchuan; Shi, Yi; Georgescu, Roxana E et al. (2015) The architecture of a eukaryotic replisome. Nat Struct Mol Biol 22:976-82
Langston, Lance D; Zhang, Dan; Yurieva, Olga et al. (2014) CMG helicase and DNA polymerase ? form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication. Proc Natl Acad Sci U S A 111:15390-5
Marzahn, Melissa R; Hayner, Jaclyn N; Finkelstein, Jeff et al. (2014) The ATP sites of AAA+ clamp loaders work together as a switch to assemble clamps on DNA. J Biol Chem 289:5537-48
Georgescu, Roxana E; Yao, Nina; Indiani, Chiara et al. (2014) Replisome mechanics: lagging strand events that influence speed and processivity. Nucleic Acids Res 42:6497-510
Georgescu, Roxana E; Langston, Lance; Yao, Nina Y et al. (2014) Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork. Nat Struct Mol Biol 21:664-70
Indiani, Chiara; O'Donnell, Mike (2013) A proposal: Source of single strand DNA that elicits the SOS response. Front Biosci (Landmark Ed) 18:312-23
Pomerantz, Richard T; Goodman, Myron F; O'Donnell, Michael E (2013) DNA polymerases are error-prone at RecA-mediated recombination intermediates. Cell Cycle 12:2558-63

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