The overarching goal of this project is to crystallize and solve the structure of a lambda integrase synaptosome multi-component complex (attL) which is inhibited at the stage of strand rearrangement to better understand how this protein precisely controls the direction of strand exchange in the process of integration/excision of DNA. In working towards this goal, we intend to try to crystallize smaller domains of this larger complex which may be more amenable to crystallization, including a tetramer of catalytic core fragments of the lambda-Integrase bound to a Holliday Junction and the full-length Integrase bound to the Holliday Junction. The proposed research involves construction of various synaptic complexes incorporating modified DNAs to trap the enzyme in the process of strand exchange. New techniques for producing long pieces of DNA (100mers) in crystallographic quantities and for assembling the multiprotein-DNA complex are proposed. To efficiently screen for crystallization conditions, we will first define conditions for the assembly of stable, homogeneous complexes. Variables include changing the length of the DNAs and the sequence of the core sites. The structure should enhance our understanding of how lambda-Int controls the direction of strand exchange within the synaptosome to dictate whether excision or integration will occur. Moreover, the project may provide new experimental approaches that are transferrable to the study of other long-ranged interactions on DNA, including communication between promoters and enhancers within transcription complexes. Eventually, this system may provide a biotechnological tool for the insertion or deletion of sequences into the genomes of higher eukaryotes to regulate gene expression or repair mutations.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM019945-03
Application #
6385048
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Cassatt, James
Project Start
1999-07-01
Project End
Budget Start
2001-07-01
Budget End
2002-06-16
Support Year
3
Fiscal Year
2001
Total Cost
$42,157
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115