Abstract

The long term goal of this proposal is to determine the enzymatic mechanisms of genetic recombination between duplex DNA molecules in Escherichia coli. The mechanism of two different recombination pathways, the RecF and RecE pathways will be studied. The basic approach that will be followed is to develop in vitro recombination systems and biochemical assays that measure genetic recombination in order to identify the biochemical intermediates and purify the proteins that are involved in genetic recombination. The mechanism of RecF pathway recombination events in vivo will be studied in detail. In particular, the effect of double-strand breaks and other types of DNA damage will be determined. RecF pathway genes will be cloned and used to overproduce and purify the proteins that they encode. The recF protein, which has already been purified, will be characterized in detail and additional oveproduction studies will concentrate on the recJ, recO, and recQ proteins. A possible role for a Holliday junction resolution enzyme in RecF pathway recombination will be investigated. In vitro systems that use crude extracts and/or partially purified proteins to catalyze RecF pathway recombination reactions will be developed and the intermediates and products that are formed in these reactions will be characterized in detail. Individual RecF pathway proteins that were not purified as part of the overproduction studies will be purified using a combination of in vitro complementation and reconstitution assays and characterized in detail. The RecE pathway recombination will be studied using the same types of methods as those described for RecF pathway reactions. Additional studies to be carried out include genetic studies to identify new genes that are required for the RecE pathway and biochemical analysis of exonuclease VIII to define the functional domain of exonuclease VIII that is required for both RecE pathway recombination and exonuclease activity. The ultimate goal of these studies will be to reconstitute RecF and RecE pathway recombination reactions with purified proteins and determine the enzymatic mechanisms of these reactions.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026017-10
Application #
3273494
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-12-01
Project End
1991-11-30
Budget Start
1987-12-01
Budget End
1988-11-30
Support Year
10
Fiscal Year
1988
Total Cost
Indirect Cost

  Institution

Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02115

  Publications

Liang, Jason; Li, Bin-Zhong; Tan, Alexander P et al. (2018) SUMO E3 ligase Mms21 prevents spontaneous DNA damage induced genome rearrangements. PLoS Genet 14:e1007250
Srivatsan, Anjana; Putnam, Christopher D; Kolodner, Richard D (2018) Analyzing Genome Rearrangements in Saccharomyces cerevisiae. Methods Mol Biol 1672:43-61
Nene, Rahul V; Putnam, Christopher D; Li, Bin-Zhong et al. (2018) Cdc73 suppresses genome instability by mediating telomere homeostasis. PLoS Genet 14:e1007170
Putnam, Christopher D; Kolodner, Richard D (2017) Pathways and Mechanisms that Prevent Genome Instability in Saccharomyces cerevisiae. Genetics 206:1187-1225
Putnam, Christopher D; Srivatsan, Anjana; Nene, Rahul V et al. (2016) A genetic network that suppresses genome rearrangements in Saccharomyces cerevisiae and contains defects in cancers. Nat Commun 7:11256
de Souza, Jorge E S; Fonseca, André F; Valieris, Renan et al. (2014) S-score: a scoring system for the identification and prioritization of predicted cancer genes. PLoS One 9:e94147
Putnam, Christopher D; Pallis, Katielee; Hayes, Tikvah K et al. (2014) DNA repair pathway selection caused by defects in TEL1, SAE2, and de novo telomere addition generates specific chromosomal rearrangement signatures. PLoS Genet 10:e1004277
Ragu, Sandrine; Dardalhon, Michèle; Sharma, Sushma et al. (2014) Loss of the thioredoxin reductase Trr1 suppresses the genomic instability of peroxiredoxin tsa1 mutants. PLoS One 9:e108123
Allen-Soltero, Stephanie; Martinez, Sandra L; Putnam, Christopher D et al. (2014) A saccharomyces cerevisiae RNase H2 interaction network functions to suppress genome instability. Mol Cell Biol 34:1521-34
Albuquerque, Claudio P; Wang, Guoliang; Lee, Nancy S et al. (2013) Distinct SUMO ligases cooperate with Esc2 and Slx5 to suppress duplication-mediated genome rearrangements. PLoS Genet 9:e1003670

Showing the most recent 10 out of 84 publications