Recombinational repair of damaged DNA and replication forks is important for genomic integrity and the prevention of mutations during growth of all organisms. E. coli serves as a valuable model system for understanding how recombinational repair occurs in more complicated eucaryotic cells. Better understanding of recombination and DNA replication can lead to new treatments for bacterial infections and cancer. Replication forks stop at stochastic, housekeeping types of DNA damage. In E. coli, evidence suggests that RecA binds to stopped forks and repairs the damage without inducing the global response to DNA damage: the SOS Response. When cells are exposed to external DNA damage, RecA also binds to these forks and repairs them. However in this case, SOS is induced. Given that RecA's binding to ssDNA at a stopped fork is critical for both repair and induction of the SOS Response, this proposal addresses why RecA induces SOS in one situation, but not the other. It is proposed that there are molecular mechanisms that allow cells to discriminate between housekeeping types of DNA damage and external DNA damage. This is important because the SOS response is an extreme response to a hazardous environment;one that should not be induced for standard growth situations. Although the actual types of DNA damage can be identical under these two situations, the key difference is in how the cell perceives the DNA damage. It is further hypothesized that the interactions between RecA and the single-stranded DNA binding protein (SSB) are critical in this decision. Three reagents have been built that reveal the position and amount of RecA, SSB and levels of SOS expression in individual cells. They use the Green Fluorescent Protein (GFP). These reagents include RecA-GFP, SSB-GFP and sulAp-gfp. Similar reagents: RecA-YFP, SSB-CFP and sulAp-rfp (yellow, cyan and red fluorescent proteins respectively) will be built so that one can measure all three at once in a single cell. This will be critical to testing the above model. The broader impact of this proposal is that one will learn how recombination and DNA replication occur temporally and spatially in the cell. This understanding will be important for developing new preventions and treatments for bacterial infections and cancers. These new reagents will also provide important molecular tools for others in the research community. PHS 398 (Rev. 09/04) Page 2 Principal Investigator/Program Director (Last, first, middle): SANDLER, STEVEN, J

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI059027-04
Application #
7595753
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Korpela, Jukka K
Project Start
2006-05-15
Project End
2011-04-30
Budget Start
2009-05-01
Budget End
2010-04-30
Support Year
4
Fiscal Year
2009
Total Cost
$254,324
Indirect Cost
Name
University of Massachusetts Amherst
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
153926712
City
Amherst
State
MA
Country
United States
Zip Code
01003
Massoni, Shawn C; Sandler, Steven J (2013) Specificity in suppression of SOS expression by recA4162 and uvrD303. DNA Repair (Amst) 12:1072-80
Massoni, Shawn C; Leeson, Michael C; Long, Jarukit Edward et al. (2012) Factors limiting SOS expression in log-phase cells of Escherichia coli. J Bacteriol 194:5325-33
Marceau, Aimee H; Bahng, Soon; Massoni, Shawn C et al. (2011) Structure of the SSB-DNA polymerase III interface and its role in DNA replication. EMBO J 30:4236-47
Long, Jarukit Edward; Massoni, Shawn C; Sandler, Steven J (2010) RecA4142 causes SOS constitutive expression by loading onto reversed replication forks in Escherichia coli K-12. J Bacteriol 192:2575-82
Long, Jarukit E; Renzette, Nicholas; Sandler, Steven J (2009) Suppression of constitutive SOS expression by recA4162 (I298V) and recA4164 (L126V) requires UvrD and RecX in Escherichia coli K-12. Mol Microbiol 73:226-39
Centore, Richard C; Leeson, Michael C; Sandler, Steven J (2009) UvrD303, a hyperhelicase mutant that antagonizes RecA-dependent SOS expression by a mechanism that depends on its C terminus. J Bacteriol 191:1429-38
Long, Jarukit Edward; Renzette, Nicholas; Centore, Richard C et al. (2008) Differential requirements of two recA mutants for constitutive SOS expression in Escherichia coli K-12. PLoS One 3:e4100
Renzette, Nicholas; Sandler, Steven J (2008) Requirements for ATP binding and hydrolysis in RecA function in Escherichia coli. Mol Microbiol 67:1347-59
Centore, Richard C; Lestini, Roxane; Sandler, Steven J (2008) XthA (Exonuclease III) regulates loading of RecA onto DNA substrates in log phase Escherichia coli cells. Mol Microbiol 67:88-101
Gruenig, Marielle C; Renzette, Nicholas; Long, Edward et al. (2008) RecA-mediated SOS induction requires an extended filament conformation but no ATP hydrolysis. Mol Microbiol 69:1165-79

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