The goal of this work is a structure-based understanding of how DNA replication is initiated. Replication initiates at multiple sites on the genome called origins of DNA replication. Specialized protein complexes bind at these sites and prepare the duplex for replication. The activity of these ensembles is tightly controlled to ensure that only one copy of the genome is made per cell cycle. The architecture, regulation and mechanisms of action of these large complexes are incompletely understood. Our work is of practical significance because regulatory changes in origin complexes contribute to human cancers. Efforts with bacterial complexes will provide much needed targets for development of novel antibiotics. We study replication initiation complexes in eukaryotes and bacteria. A large body of work has described components of origin complexes and details of how they operate. What is missing, however, in any of these cases, is a three-dimensional view of the initiation machine to guide our understanding of how these components work together. The overall goal of our research is to provide such views. The current proposal incorporates three complementary approaches that focus on several components involved in origin recognition and DNA unwinding.
Aim #1 is directed at several bacterial helicaseloader complexes, whose structure and function can be dissected at a sophisticated level and will inform our understanding of eukaryotic complexes.
Aim #2 is addressed at obtaining and analyzing the complete ORC ensemble.
Aim #3 exploits our recent identification of a unique bacterial MCM complex, which is more tractable, but should still provide a faithful model for its eukaryotic counterparts.

Public Health Relevance

Cells prepare for the next round of DNA replication by depositing macromolecular assemblies onto sites on the genome termed origins of DNA replication. Emerging evidence suggests that these DNA replication initiation assemblies represent a rich collection of molecular targets against which to design novel therapeutics. This pathway is not well represented in efforts to discover anti-cancer agents. Lack of useful structural information on relevant molecular targets has stymied efforts at structure-guided drug design. Our structure determination efforts will advance efforts to discover novel anti-cancer agents. Also, our work with bacterial complexes will provide much needed targets for development of novel antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM084162-01A2
Application #
7652564
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Santangelo, George M
Project Start
2009-04-01
Project End
2013-03-31
Budget Start
2009-04-01
Budget End
2010-03-31
Support Year
1
Fiscal Year
2009
Total Cost
$308,400
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Pakotiprapha, Danaya; Jeruzalmi, David (2013) Small-angle X-ray scattering reveals architecture and A?B? stoichiometry of the UvrA-UvrB DNA damage sensor. Proteins 81:132-9
Pakotiprapha, Danaya; Samuels, Martin; Shen, Koning et al. (2012) Structure and mechanism of the UvrA-UvrB DNA damage sensor. Nat Struct Mol Biol 19:291-8
Samuels, Martin; Gulati, Gaurav; Shin, Jae-Ho et al. (2009) A biochemically active MCM-like helicase in Bacillus cereus. Nucleic Acids Res 37:4441-52