The continuous movement of the DNA replication fork requires priming at the lagging strand to make each Okazaki fragment, as well as a coordinated action of primases and the replicative DNA polymerase. The replication origin of the single-stranded DNA (ssDNA) bacteriophage G4 (G4ori) is a simple system of priming for DNA replication; it needs only the E. coli primase (dnaG) and the single-stranded DNA binding protein (SSB). G4ori consists of three stem-loops located immediately on the 5' side of the initiation trinucleotide 5'-CTG-3' and, upon saturated binding by SSB tetramers, will form a unique structure recognizable by dnaG primase. The three domains of the modular primase then bind the G4ori initiation site cooperatively to synthesize the primer RNA. A 278 nucleotide fragment of G4ori (G4ori278), together with two dnaG primases and four SSB tetramers, can form a minimal active priming complex suitable for structural investigations. The continuous movement of the replication fork also requires recombinational repair at sites of DNA damage and recFOR complexes of the recF recombination pathway are mediators of replication and recombination. Structural information of these replication and recombination complexes will help us understand the function of the replication machineries. Toward this goal, we propose three sets of experiments: 1) To determine the X-ray structures of the two-component complex of SSB-G4ori278 and a complex of SSB with a fragment of the lacZ gene. These structures will help us understand the structural formation of the functional G4ori, as well as SSB's modes of ssDNA-binding. 2) To determine the X-ray structure of the three-component complex of primase-SSB-G4ori278, which will reveal details of the primase- G4ori interaction, as well as the primase-SSB interaction. 3) To determine the X-ray structure of recR and to crystallize other proteins, as well as complexes in the recF pathway. Obtaining the structural information of these proteins and complexes will serve as an important step in understanding the recombinational repair. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM070841-02
Application #
7010683
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Lewis, Catherine D
Project Start
2005-02-01
Project End
2009-01-31
Budget Start
2006-02-01
Budget End
2007-01-31
Support Year
2
Fiscal Year
2006
Total Cost
$365,356
Indirect Cost
Name
New York University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
121911077
City
New York
State
NY
Country
United States
Zip Code
10016
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