Our long range goal continues to be a detailed understanding of the molecular mechanisms involved in site-specific recombination, including the associated higher-order structures and protein-DNA and protein-protein interactions. As a model we are studying the Int-dependent recombination pathways of the lambdoid phages lambda, phi80, and P22. The major portion of our effort is directed toward the A system but the information we have been obtaining about the other two systems is proving to be very useful at both the experimental and conceptual levels. The experiments proposed in this application are intended to complement studies being carried out in other laboratories. They are based primarily on the following three groups of results we obtained during the previous project period: a) the Int protein has a unique dual DNA recognition capacity that is specified by two autonomous domains and a topoisomerase active site that is highly conserved in more than 15 other proteins; b) there is an additional host-encoded accessory protein (FIS) that ties lambda recombination to cellular physiology and functions, along with the other accessory proteins (IHF and Xis), by inducing very extreme bends in DNA; c) there are numerous and complex interactions among the four kinds of proteins bound at the 15 binding sites on att DNA. Four specific questions are addressed in this proposal: 1)What structural features of the two-domain Int protein are critical for its function as a site-specific recombinase? 2)How do the recombinogenic structures and mechanisms utilize the accessory proteins IHF, FIS and Xis? 3)What are the short-range and long-range interactions that define the higher order structure of the recombinogenic complexes and determine the efficiency and specificity of recombination? 4)How is the extreme directionality of recombination achieved?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI013544-17
Application #
3480758
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Project Start
1977-09-01
Project End
1995-07-31
Budget Start
1993-08-01
Budget End
1994-07-31
Support Year
17
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Brown University
Department
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
Tirumalai, R S; Kwon, H J; Cardente, E H et al. (1998) Recognition of core-type DNA sites by lambda integrase. J Mol Biol 279:513-27
Nunes-Duby, S E; Kwon, H J; Tirumalai, R S et al. (1998) Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26:391-406
Tirumalai, R S; Healey, E; Landy, A (1997) The catalytic domain of lambda site-specific recombinase. Proc Natl Acad Sci U S A 94:6104-9
Kwon, H J; Tirumalai, R; Landy, A et al. (1997) Flexibility in DNA recombination: structure of the lambda integrase catalytic core. Science 276:126-31
Nunes-Duby, S E; Yu, D; Landy, A (1997) Sensing homology at the strand-swapping step in lambda excisive recombination. J Mol Biol 272:493-508
Azaro, M A; Landy, A (1997) The isomeric preference of Holliday junctions influences resolution bias by lambda integrase. EMBO J 16:3744-55
Tirumalai, R S; Pargellis, C A; Landy, A (1996) Identification and characterization of the N-ethylmaleimide-sensitive site in lambda-integrase. J Biol Chem 271:29599-604
Zahn, K; Landy, A (1996) Modulation of lambda integrase synthesis by rare arginine tRNA. Mol Microbiol 21:69-76
Nunes-Duby, S E; Azaro, M A; Landy, A (1995) Swapping DNA strands and sensing homology without branch migration in lambda site-specific recombination. Curr Biol 5:139-48
Nunes-Duby, S E; Smith-Mungo, L I; Landy, A (1995) Single base-pair precision and structural rigidity in a small IHF-induced DNA loop. J Mol Biol 253:228-42

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