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Jh, New Stephen Charles Kowalczykowski
Published in
Journal of Biological Chemistry
Rad52 protein plays a central role in double strand break repair and homologous recombination in Saccharomyces cerevisiae. We have identified a new mechanism by which Rad52 protein stimulates Rad51 protein-promoted DNA strand exchange. This function of Rad52 protein is revealed when subsaturating amounts (relative to the single-stranded DNA concent...
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P, Cejka Jl, Plank Cc, Dombrowski Stephen Charles Kowalczykowski
Published in
Molecular Cell
Genetic evidence indicates that Saccharomyces cerevisiae Sgs1, Top3, and Rmi1 resolve topologically linked intermediates arising from DNA replication and recombination. Using purified proteins, we show that Sgs1, Top3, Rmi1, and replication protein A (RPA) coordinate catenation and decatenation of dsDNA through sequential passage of single strands ...
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A, Alexeev A, Mazin Stephen Charles Kowalczykowski
Published in
Natural Structural Biology
In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, Rad52, Rad55 and Rad57 proteins. In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein. Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dep...
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Y, Wu N, Kantake T, Sugiyama Stephen Charles Kowalczykowski
Published in
Journal of Biological Chemistry
In Saccharomyces cerevisiae, Rad52 protein plays an essential role in the repair of DNA double-stranded breaks (DSBs). Rad52 and its orthologs possess the unique capacity to anneal single-stranded DNA (ssDNA) complexed with its cognate ssDNA-binding protein, RPA. This annealing activity is used in multiple mechanisms of DSB repair: single-stranded ...
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Stephen Charles Kowalczykowski
Published in
Annual Review of Biophysics and Bioengineering
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Av, Nimonkar Stephen Charles Kowalczykowski
Published in
Cell Cycle
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En, Zaitsev Stephen Charles Kowalczykowski
Published in
Journal of Molecular Biology
We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4 ,6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the R...
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T, Sugiyama Stephen Charles Kowalczykowski
Published in
Journal of Biological Chemistry
The Rad51 nucleoprotein filament mediates DNA strand exchange, a key step of homologous recombination. This activity is stimulated by replication protein A (RPA), but only when RPA is introduced after Rad51 nucleoprotein filament formation. In contrast, RPA inhibits Rad51 nucleoprotein complex formation by prior binding to single-stranded DNA (ssDN...
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Stephen Charles Kowalczykowski Ra, Krupp
Published in
Proceedings of the National Academy of Sciences
DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange...
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Pg, Mitsis Stephen Charles Kowalczykowski Ir, Lehman
Published in
Biochemistry
We describe the purification to near homogeneity of a single-stranded DNA binding protein from 0-18-h embryos of Drosophila melanogaster. Drosophila SSB (D-SSB) is a heterotrimer with subunits of molecular weight of 70,000, 30,000, and 8000. It has a Stokes radius of 48.6 +/- 2 A and s20,w = 5.0 +/- 0.2 S. The interaction of D-SSB with ssDNA was ex...
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Jp, Menetski Stephen Charles Kowalczykowski
Published in
Biochemistry
The Escherichia coli recA protein has been shown to hydrolyze several nucleoside triphosphates in the presence of ssDNA. The substitution of dATP for rATP has significant effects on various recA protein biochemical properties. In the presence of dATP, recA protein can invade more secondary structure in native ssDNA than it can in the presence of rA...
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E, Cannavo P, Cejka Stephen Charles Kowalczykowski
Published in
Proceedings of the National Academy of Sciences
Homologous recombination is a major pathway for repair of DNA double-strand breaks. This repair process is initiated by resection of the 5′-terminated strand at the break site. In yeast, resection is carried out by three nucleolytic complexes: Mre11-Rad50-Xrs2, which functions at the initial step and also stimulates the two processive pathways, Sgs...
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Sd, Lauder Stephen Charles Kowalczykowski
Published in
Journal of Biological Chemistry
The apparent DNA site size obtained from an assay monitoring the ATPase activity of Escherichia coli recA protein (n = 3.5) differs from that determined from a direct DNA binding assay (n = 7) done under identical conditions. Investigation of this discrepancy indicates that at a DNA:protein ratio of 3.5:1, one-half of the recA protein population is...
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T, Sugiyama Stephen Charles Kowalczykowski Y, Wu Js, Siino
Published in
Journal of Biological Chemistry
We examined the double-stranded DNA (dsDNA) binding preference of the Saccharomyces cerevisiae Rad52 protein and its homologue, the Rad59 protein. In nuclease protection assays both proteins protected an internal sequence and the dsDNA ends equally well. Similarly, using electrophoretic mobility shift assays, we found the affinity of both Rad52 and...
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Dg, Anderson Stephen Charles Kowalczykowski
Published in
Cell
Double-stranded DNA break repair and homologous recombination in E. coli are initiated by the RecBCD enzyme, which unwinds and simultaneously degrades DNA from a double-stranded DNA end. This process is stimulated by cis-acting DNA elements, known as chi sites. Using both in vitro pairing and nuclease protection assays, we demonstrate that the tran...
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Jp, Menetski Stephen Charles Kowalczykowski
Published in
Journal of Biological Chemistry
We have analyzed the transfer kinetics of recA protein from one polynucleotide to another by monitoring the change in fluorescence of a modified single-stranded M13 DNA, referred to as etheno M13 DNA, that accompanies recA protein dissociation. The observed rate of transfer is dependent on the concentration of competitor polynucleotide, polythymidy...
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B, Rad Al, Forget Rj, Baskin Stephen Charles Kowalczykowski
Published in
Proceedings of the National Academy of Sciences
DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules...
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Fg, Harmon Jp, Brockman Stephen Charles Kowalczykowski
Published in
Journal of Biological Chemistry
Together, RecQ helicase and topoisomerase III (Topo III) of Escherichia coli comprise a potent DNA strand passage activity that can catenate covalently closed DNA (Harmon, F. G., DiGate, R. J., and Kowalczykowski, S. C. (1999) Mol. Cell 3, 611-620). Here we directly assessed the structure of the catenated DNA species formed by RecQ helicase and Top...
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Stephen Charles Kowalczykowski
Published in
Cold Spring Harbor Perspectives in Biology
Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those...
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Da, Dixon Stephen Charles Kowalczykowski
Published in
Journal of Biological Chemistry
Genetic recombination occurring in wild type Escherichia coli is stimulated at DNA sequences known as chi sites, 5 -GCTGGTGG-3 . In vitro, homologous pairing between duplex DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is stimulated by the presence of a chi sequence in the donor linear double-stranded DNA. We show that this stimu...