The enzymology of homologous pairing and strand exchange promoted by E. coli recA protein's unique. Whereas replication and transcription involve multi-enzyme complexes built from large sets of different polypeptide chains, recA protein polymerizes on single-stranded DNA to treat a multimolecular and helical enzymatic machine that is capable of promoting homologous pairing and strand exchange. Only one other protein, E. coli single-stranded DNA binding protein (SSB) is known to participate directly with recA protein in pairing and strand exchange, but other enzymes must create the broken DNA substrates on which recA protein can act, and must resolve the intermediates created by recA protein. The long term objective of the research proposed here is to define enzymatically the chain of events that constitute the major pathway of homologous recombination in E. coli. One means to that end, and an intermediate goal is the complete reconstitution of homologous recombination in vitro from purified enzymes. Our short range goals are: 1) to set up and develop the assay based on the packaging of lambda DNA in order to detect sensitively recombination of DNA molecules in crude extracts of E. coli, 2) through the use of particular constructs of phage lambda for crosses in known mutants of E. coli, to elucidate as far as possible the set of enzymes that are required in vivo for homologous recombination controlled by the principal pathway of recombination, the recABCD pathway, 3) to use the packaging assay (item I above and the in vivo observations (item 2 above) to develop an in vitro system starting from crude extracts. as a means to purify and identify a minimal set of enzymes required for homologous recombination, 4) to study mutational changes in the entire set of known recombine activities of recA protein purified from new recA mutants in order to analyze the mechanism of homologous pairing and strand exchange, and 5) to analyze further the topological requirements, the changes in DNA conformation, and the intermediates that are involved in the homologous pairing of naked duplex DNA with the helical recA nucleoprotein filament. Homologous recombination, one of the keystones of genetics, is a subject of fundamental interest and importance. The use of homologous recombination to """"""""target"""""""" genes to specific chromosomal sites in mammalian cells may significantly expand the genetic tools available to study mammalian cells and may eventually provide one of the means necessary to accomplish gene therapy.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033504-06
Application #
3283292
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-05-01
Project End
1994-04-30
Budget Start
1989-05-01
Budget End
1990-04-30
Support Year
6
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Noirot, Philippe; Gupta, Ravindra C; Radding, Charles M et al. (2003) Hallmarks of homology recognition by RecA-like recombinases are exhibited by the unrelated Escherichia coli RecT protein. EMBO J 22:324-34
Passy, S I; Yu, X; Li, Z et al. (1999) Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins. Proc Natl Acad Sci U S A 96:4279-84
Chiu, S K; Low, K B; Yuan, A et al. (1997) Resolution of an early RecA-recombination intermediate by a junction-specific endonuclease. Proc Natl Acad Sci U S A 94:6079-83
Plug, A W; Xu, J; Reddy, G et al. (1996) Presynaptic association of Rad51 protein with selected sites in meiotic chromatin. Proc Natl Acad Sci U S A 93:5920-4
Kurumizaka, H; Aihara, H; Ikawa, S et al. (1996) A possible role of the C-terminal domain of the RecA protein. A gateway model for double-stranded DNA binding. J Biol Chem 271:33515-24
Reddy, G; Burnett, B; Radding, C M (1995) Uptake and processing of duplex DNA by RecA nucleoprotein filaments: insights provided by a mixed population of dynamic and static intermediates. Biochemistry 34:10194-204
Rao, B J; Radding, C M (1995) RecA protein mediates homologous recognition via non-Watson-Crick bonds in base triplets. Philos Trans R Soc Lond B Biol Sci 347:5-12
Ashley, T; Plug, A W; Xu, J et al. (1995) Dynamic changes in Rad51 distribution on chromatin during meiosis in male and female vertebrates. Chromosoma 104:19-28
Rao, B J; Chiu, S K; Bazemore, L R et al. (1995) How specific is the first recognition step of homologous recombination? Trends Biochem Sci 20:109-13
Haaf, T; Golub, E I; Reddy, G et al. (1995) Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes. Proc Natl Acad Sci U S A 92:2298-302

Showing the most recent 10 out of 34 publications