Papers by Stephen Van Komen
This article cites 61 articles, 35 of which can be accessed free
... 昭57843587，2002 戶口n@ed@nUS且． Functional Cross-talk among Rad51, Rad54, A in Heteroduplex DNA J... more ... 昭57843587，2002 戶口n@ed@nUS且． Functional Cross-talk among Rad51, Rad54, A in Heteroduplex DNA Joint Formation* a血dRepHcationPro柏i几 Received for publication, June 12, 2002, and in revised form, September 3, 2002 Published, JBC Papers in Press ...
Progress in Nucleic Acid Research and Molecular Biology, 2003
DNA double-strand breaks (DSBs) pose a special challenge for cells in the maintenance of genome s... more DNA double-strand breaks (DSBs) pose a special challenge for cells in the maintenance of genome stability. In eukaryotes, the removal of DSBs is mediated by two major pathways: homologous recombination (HR) and nonhomologous endjoining (NHEJ). Capitalizing on existing genetic frameworks, biochemical reconstitution studies have begun to yield insights into the mechanistic underpinnings of these DNA repair reactions.
Molecular Cell, 2000
DNA homolog, either the sister chromatid or homologous chromosome, to form a heteroduplex DNA joi... more DNA homolog, either the sister chromatid or homologous chromosome, to form a heteroduplex DNA joint called D loop. The recombination event is completed by
Methods in enzymology, 2006
Homologous recombination is an important means of eliminating DNA double strand breaks from chrom... more Homologous recombination is an important means of eliminating DNA double strand breaks from chromosomes. The homologous recombination reaction is mediated by the Rad51 recombinase, which requires a number of ancillary factors for maximal efficiency. The development of purification procedures and biochemical assays for yeast Rad51 and other yeast recombination proteins has allowed investigators to begin dissecting the hierarchy of physical and functional interactions among these protein factors that govern the integrity of the homologous recombination machinery. The biochemical studies done with yeast recombination factors have helped formulate conceptual frameworks to guide similar endeavors in other eukaryotes, including humans. Continuing efforts with reconstituted systems that comprise yeast factors will undoubtedly continue to provide insights into the mechanistic intricacy of the homologous recombination machinery.
Molecular and cellular biology, 2005
Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Althou... more Werner syndrome, caused by mutations of the WRN gene, mimics many changes of normal aging. Although roles for WRN protein in DNA replication, recombination, and telomere maintenance have been suggested, the pathology of rapidly dividing cells is not a feature of Werner syndrome. To identify cellular events that are specifically vulnerable to WRN deficiency, we used RNA interference (RNAi) to knockdown WRN or BLM (the RecQ helicase mutated in Bloom syndrome) expression in primary human fibroblasts. Withdrawal of WRN or BLM produced accelerated cellular senescence phenotype and DNA damage response in normal fibroblasts, as evidenced by induction of gammaH2AX and 53BP1 nuclear foci. After WRN depletion, the induction of these foci was seen most prominently in nondividing cells. Growth in physiological (3%) oxygen or in the presence of an antioxidant prevented the development of the DNA damage foci in WRN-depleted cells, whereas acute oxidative stress led to inefficient repair of the le...
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 2000
The budding yeast Saccharomyces cerevisiae has been an excellent genetic and biochemical model fo... more The budding yeast Saccharomyces cerevisiae has been an excellent genetic and biochemical model for our understanding of homologous recombination. Central to the process of homologous recombination are the products of the RAD52 epistasis group of genes, whose functions we now know include the nucleolytic processing of DNA double-stand breaks, the ability to conduct a DNA homology search, and the capacity to promote the exchange of genetic information between homologous regions on recombining chromosomes. It is also clear that the basic functions of the RAD52 group of genes have been highly conserved among eukaryotes. Disruption of this important process causes genomic instability, which can result in a number of unsavory consequences, including tumorigenesis and cell death.
Nature, 2003
mice with or without the C57Bl/Ka-Ly5.2 recipient bone marrow cells 1. Reconstitution of donor (L... more mice with or without the C57Bl/Ka-Ly5.2 recipient bone marrow cells 1. Reconstitution of donor (Ly5.1) myeloid and lymphoid cells was monitored by staining blood cells with antibodies against Ly5.1, CD3, B220, Mac-1 and Gr-1. The secondary bone marrow transplant was performed with 10 7 whole bone marrow cells from mice reconstituted with Bmi-1 þ/þ or Bmi-1 2/2 fetal liver cells. Retroviral gene transfer of HSCs Mouse stem cell viruses expressing mouse p16 Ink4a or p19 Arf cDNAs together with GFP were produced using Phoenix ecotropic packaging cells 28. Infection of HSCs was done as described 29 except that three cycles of infections were performed. After 48 h, single GFP-positive cells were sorted into a 96-well plate containing 100 ml HSC medium 29 and grown for 7 days. Each well was scored for the presence of GFP-positive cells by observation with a fluorescence microscope.
Molecular Cell, 2003
In the mouse, a homozygous null allele of RAD51 leads to embryonic lethality (Tsuzuki et al., 199... more In the mouse, a homozygous null allele of RAD51 leads to embryonic lethality (Tsuzuki et al., 1996), and muta
Molecular and Cellular Biology, 2006
Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here... more Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, ...
Journal of Biological Chemistry, 2004
In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the... more In eukaryotes, Rad51 and Rad54 functionally cooperate to mediate homologous recombination and the repair of damaged chromosomes by recombination. Rad51, the eukaryotic counterpart of the bacterial RecA recombinase, forms filaments on single-stranded DNA that are capable of pairing the bound DNA with a homologous double-stranded donor to yield joint molecules. Rad54 enhances the homologous DNA pairing reaction, and this stimulatory effect involves a physical interaction with Rad51. Correspondingly, the ability of Rad54 to hydrolyze ATP and introduce superhelical tension into covalently closed circular plasmid DNA is stimulated by Rad51. By controlled proteolysis, we show that the amino-terminal region of yeast Rad54 is rather unstructured. Truncation mutations that delete the N-terminal 113 or 129 amino acid residues of Rad54 attenuate or ablate physical and functional interactions with Rad51 under physiological ionic strength, respectively. Surprisingly, under less stringent conditi...
Journal of Biological Chemistry, 2002
Journal of Biological Chemistry, 2004
Yeast RAD54 gene, a member of the RAD52 epistasis group, plays an important role in homologous re... more Yeast RAD54 gene, a member of the RAD52 epistasis group, plays an important role in homologous recombination and DNA double strand break repair. Rad54 belongs to the Snf2/Swi2 protein family, and it possesses a robust DNA-dependent ATPase activity, uses free energy from ATP hydrolysis to supercoil DNA, and cooperates with the Rad51 recombinase in DNA joint formation. There are two RAD54-homologous genes in human cells, hRAD54 and RAD54B. Mutations in these human genes have been found in tumors. These tumor-associated mutations map to conserved regions of the hRad54 and hRad54B proteins. Here we introduced the equivalent mutations into the Saccharomyces cerevisiae RAD54 gene in an effort to examine the functional consequences of these gene changes. One mutant, rad54 G484R, showed sensitivity to DNA-damaging agents and reduced homologous recombination rates, indicating a loss of function. Even though the purified rad54 G484R mutant protein retained the ability to bind DNA and interact with Rad51, it was nearly devoid of ATPase activity and was similarly defective in DNA supercoiling and D-loop formation. Two other mutants, rad54 N616S and rad54 D442Y, were not sensitive to genotoxic agents and behaved like the wild type allele in homologous recombination assays. Consistent with the mild phenotype associated with the rad54 N616S allele, its encoded protein was similar to wild type Rad54 protein in biochemical attributes. Because dysfunctional homologous recombination gives rise to genome instability, our results are consistent with the premise that tumor-associated mutations in hRad54 and Rad54B could contribute to the tumor phenotype or enhance the genome instability seen in tumor cells.
Journal of Biological Chemistry, 2004
Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genoto... more Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G 2 /M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded(ss)DNAdependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1⌬ or rad54⌬ mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.
Journal of Biological Chemistry, 2002
Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologou... more Human Rad51 (hRad51) and Rad54 proteins are key members of the RAD52 group required for homologous recombination. We show an ability of hRad54 to promote transient separation of the strands in duplex DNA via its ATP hydrolysis-driven DNA supercoiling function. The ATPase, DNA supercoiling, and DNA strand opening activities of hRad54 are greatly stimulated through an interaction with hRad51. Importantly, we demonstrate that hRad51 and hRad54 functionally cooperate in the homologous DNA pairing reaction that forms recombination DNA intermediates. Our results should provide a biochemical model for dissecting the role of hRad51 and hRad54 in recombination reactions in human cells.
Journal of Biological Chemistry, 1999
Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key interme... more Saccharomyces cerevisiae RAD54 gene functions in the formation of heteroduplex DNA, a key intermediate in recombination processes. Rad54 is monomeric in solution, but forms a dimer/oligomer on DNA. Rad54 dimer/oligomer alters the conformation of the DNA double helix in an ATP-dependent manner, as revealed by a change in the DNA linking number in a topoisomerase I-linked reaction. DNA conformational alteration does not occur in the presence of non-hydrolyzable ATP analogues, nor when mutant rad54 proteins defective in ATP hydrolysis replace Rad54. Accordingly, the Rad54 ATPase activity is shown to be required for biological function in vivo and for promoting Rad51-mediated homologous DNA pairing in vitro. Taken together, the results are consistent with a model in which a Rad54 dimer/oligomer promotes nascent heteroduplex joint formation via a specific interaction with Rad51 protein and an ability to transiently unwind duplex DNA.
Journal of Biological Chemistry, 2005
The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of el... more The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene. Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway. The Mph1 protein contains the seven conserved motifs of the superfamily 2 (SF2) family of nucleic acid unwinding enzymes. Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination. To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity. We show that Mph1 has a robust ATPase function that requires single-stranded DNA for activation. Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1. We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3 to 5 polarity with respect to the DNA strand on which this protein translocates. The DNA helicase activity of Mph1 is enhanced by the heterotrimeric single-stranded DNA binding protein replication protein A. These results, thus, establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance.
Journal of Biological Chemistry, 2004
The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular respon... more The Saccharomyces cerevisiae Rad50-Mre11-Xrs2 complex plays a central role in the cellular response to DNA double strand breaks. Rad50 has a globular ATPase head domain with a long coiled-coil tail. DNA binding by Rad50 is ATP-dependent and the Rad50-Mre11-Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP. Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double strand break repair by a combination of genetic and biochemical approaches. Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation. The Walker A mutations also cause a deficiency in non-homologous end-joining. As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities. Although the DNA endbridging activity of the Rad50-Mre11-Xrs2 complex is ATP-independent, the end-bridging activity of complexes containing the rad50 Walker A mutant proteins is salt-sensitive. These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging. DNA double strand breaks (DSBs) 1 arise from a variety of sources including normal physiological programs, such as * This work was supported by Grants R01 GM47251
Journal of Biological Chemistry, 2003
Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage... more Saccharomyces cerevisiae SRS2 encodes an ATP-dependent DNA helicase that is needed for DNA damage checkpoint responses and that modulates the efficiency of homologous recombination. Interestingly, strains simultaneously mutated for SRS2 and a variety of DNA repair genes show low viability that can be overcome by inactivating homologous recombination, thus implicating inappropriate recombination as the cause of growth impairment in these mutants. Here, we report on our biochemical characterization of the ATPase and DNA helicase activities of Srs2. ATP hydrolysis by Srs2 occurs efficiently only in the presence of DNA, with ssDNA being considerably more effective than dsDNA in this regard. Using homopolymeric substrates, the minimal DNA length for activating ATP hydrolysis is found to be 5 nucleotides, but a length of 10 nucleotides is needed for maximal activation. In its helicase action, Srs2 prefers substrates with a 3 ss overhang, and ϳ10 bases of 3 overhanging DNA is needed for efficient targeting of Srs2 to the substrate. Even though a 3 overhang serves to target Srs2, under optimized conditions blunt-end DNA substrates are also dissociated by this protein. The ability of Srs2 to unwind helicase substrates with a long duplex region is enhanced by the inclusion of the singlestrand DNA-binding factor replication protein A.
Journal of Biological Chemistry, 2003
In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires ... more In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecAlike recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a chromatinremodeling enzyme that promotes homologous DNA pairing events within the context of chromatin.
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Papers by Stephen Van Komen