Stephen Charles Kowalczykowski

Rad52 protein has a second stimulatory role in DNA strand exchange that complements replication protein-A function.

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...

Decatenation of DNA by the S. cerevisiae Sgs1-Top3-Rmi1 and RPA complex: a mechanism for disentangling chromosomes.

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 ...

Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament.

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...

Rad51 protein controls Rad52-mediated DNA annealing.

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 ...

Biochemistry of genetic recombination: energetics and mechanism of DNA strand exchange.

Published in Annual Review of Biophysics and Bioengineering

Second-end DNA capture in double-strand break repair: how to catch a DNA by its tail.

Published in Cell Cycle

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein.

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...

Rad52 protein associates with replication protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement ...

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...

DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transductio...

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...

A single-stranded DNA binding protein from Drosophila melanogaster: characterization of the heterotrimeric protein and i...

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...

Enhancement of Escherichia coli RecA protein enzymatic function by dATP.

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...

Relationship of DNA degradation by Saccharomyces cerevisiae exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs...

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...

Asymmetry in the recA protein-DNA filament.

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...

The DNA binding preference of RAD52 and RAD59 proteins: implications for RAD52 and RAD59 protein function in homologous ...

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...

The translocating RecBCD enzyme stimulates recombination by directing RecA protein onto ssDNA in a chi-regulated manner....

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...

Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the tran...

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...

Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive...

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...

RecQ helicase stimulates both DNA catenation and changes in DNA topology by topoisomerase III.

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...

An Overview of the Molecular Mechanisms of Recombinational DNA Repair.

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...

Role of the Escherichia coli recombination hotspot, chi, in RecABCD-dependent homologous pairing.

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...