Kowalczykowski Lab - UC Davis
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1 News 192 Publications 19 Members

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

Rad, Behzad Forget, Anthony L Baskin, Ronald J Kowalczykowski, Stephen C

Published in Proceedings of the National Academy of Sciences of the United States of America

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...

MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex.

Lee, Kyung Yong Im, Jun-Sub Shibata, Etsuko Park, Jonghoon Handa, Naofumi Kowalczykowski, Stephen C Dutta, Anindya

Published in Nature communications

MCM8-9 complex is required for homologous recombination (HR)-mediated repair of double-strand breaks (DSBs). Here we report that MCM8-9 is required for DNA resection by MRN (MRE11-RAD50-NBS1) at DSBs to generate ssDNA. MCM8-9 interacts with MRN and is required for the nuclease activity and stable association of MRN with DSBs. The ATPase motifs of M...

DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate.

Liu, Bian Baskin, Ronald J Kowalczykowski, Stephen C

Published in Nature

Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing...

Pif1 family helicases suppress genome instability at G-quadruplex motifs

Paeschke, Katrin Bochman, Matthew L. Garcia, P. Daniela Cejka, Petr Friedman, Katherine L. Kowalczykowski, Stephen C. Zakian, Virginia A.

Published in Nature

Pif1 family DNA helicases are highly conserved, from bacteria to humans. Virginia Zakian and colleagues show that in yeast, human PIF1 can unwind a four-stranded structure known as a G-quadruplex. In doing so, it suppresses the genomic instability that arises at such structures. The ability of the human protein to complement in yeast demonstrates t...

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

Cejka, Petr Plank, Jody L Dombrowski, Christopher C Kowalczykowski, Stephen C

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 ...

Polyubiquitinated PCNA recruits the ZRANB3 translocase to maintain genomic integrity after replication stress.

Ciccia, Alberto Nimonkar, Amitabh V Hu, Yiduo Hajdu, Ildiko Achar, Yathish Jagadheesh Izhar, Lior Petit, Sarah A Adamson, Britt Yoon, John C Kowalczykowski, Stephen C ...

Published in Molecular cell

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, ...

BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair.

Nimonkar, Amitabh V Genschel, Jochen Kinoshita, Eri Polaczek, Piotr Campbell, Judith L Wyman, Claire Modrich, Paul Kowalczykowski, Stephen C

Published in Genes & development

Repair of dsDNA breaks requires processing to produce 3'-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, ...

Single-molecule imaging brings Rad51 nucleoprotein filaments into focus.

Al, Forget Sc, Kowalczykowski

Published in Trends in Cell Biology

The Rad51 protein is essential for DNA repair by homologous recombination. After DNA damage, Rad51 localizes to nuclear foci that represent sites of DNA repair in vivo. In vitro, Rad51 self-assembles on single- or double-stranded DNA to form a nucleoprotein filament. Recently, the merging of innovative single-molecule techniques with ensemble metho...

The full-length Saccharomyces cerevisiae Sgs1 protein is a vigorous DNA helicase that preferentially unwinds holliday ju...

Cejka, Petr Kowalczykowski, Stephen C

Published in The Journal of biological chemistry

The highly conserved RecQ family of DNA helicases has multiple roles in the maintenance of genome stability. Sgs1, the single RecQ homologue in Saccharomyces cerevisiae, acts both early and late during homologous recombination. Here we present the expression, purification, and biochemical analysis of full-length Sgs1. Unlike the truncated form of S...

Watching Individual Proteins Acting on Single Molecules of DNA

Amitani, Ichiro Liu, Bian Dombrowski, Christopher C. Baskin, Ronald J. Kowalczykowski, Stephen C.

Published in Methods in Enzymology

In traditional biochemical experiments, the behavior of individual proteins is obscured by ensemble averaging. To better understand the behavior of proteins that bind to and/or translocate on DNA, we have developed instrumentation that uses optical trapping, microfluidic solution delivery, and fluorescent microscopy to visualize either individual p...

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