Biochemical Basis of the Constitutive Coprotease Activity of RecA P67W Protein

Mirshad, Julie K.; Kowalczykowski, Stephen C.

doi:10.1021/bi027232q

Cited by 8 publications

(6 citation statements)

References 46 publications

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“…In these experiments, ATP and SSB were added to circular ssDNA first to allow SSB to bind and coat the circular ssDNA, and then RecA protein was added to initiate ATP hydrolysis. A long lag was observed before a steady state rate of ATP hydrolysis was reached for all proteins, consistent with the fact that RecA protein has a quite limited capacity to displace SSB from ssDNA [40–43]. As conditions for ATP hydrolysis were not optimal, k cat was not calculated for these experiments.…”

Section: Resultsmentioning

confidence: 72%

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

et al. 2015

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Among strains of Escherichia coli that have evolved to survive extreme exposure to ionizing radiation, mutations in the recA gene are prominent and contribute substantially to the acquired phenotype. Changes at amino acid residue 276, D276A and D276N, occur repeatedly and in separate evolved populations. RecA D276A and RecA D276N exhibit unique adaptations to an environment that can require the repair of hundreds of double strand breaks. These two RecA protein variants (a) exhibit a faster rate of filament nucleation on DNA, as well as a slower extension under at least some conditions, leading potentially to a distribution of the protein among a higher number of shorter filaments, (b) promote DNA strand exchange more efficiently in the context of a shorter filament, and (c) are markedly less inhibited by ADP. These adaptations potentially allow RecA protein to address larger numbers of double strand DNA breaks in an environment where ADP concentrations are higher due to a compromised cellular metabolism.

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Section: Resultsmentioning

confidence: 72%

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

et al. 2015

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“…However, the ability of the E38K variant to suppress the inability of recA K72R to induce the SOS response is likely related to its capacity to form extended filaments, as exhibited by a constitutive SOS phenotype. Many other recA mutants have been shown to have an SOS C phenotype (McCall et al ., 1987; Lavery and Kowalczykowski, 1992; Tateishi et al ., 1992; Ennis et al ., 1995; McGrew and Knight, 2003; Mirshad and Kowalczykowski, 2003; O'Reilly and Kreuzer, 2004). It is plausible that other recA variants that confer an SOS C phenotype may be able to suppress the SOS‐deficient phenotype of recA K72R, but it is also clear that not all of them do.…”

Section: Discussionmentioning

confidence: 99%

RecA‐mediated SOS induction requires an extended filament conformation but no ATP hydrolysis

Gruenig

Renzette

Long

et al. 2008

Molecular Microbiology

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SummaryThe Escherichia coli SOS response to DNA damage is modulated by the RecA protein, a recombinase that forms an extended filament on single-stranded DNA and hydrolyzes ATP. The RecA K72R (recA2201) mutation eliminates the ATPase activity of RecA protein. The mutation also limits the capacity of RecA to form long filaments in the presence of ATP. Strains with this mutation do not undergo SOS induction in vivo. We have combined the K72R variant of RecA with another mutation, RecA E38K (recA730). In vitro, the double mutant RecA E38K/K72R (recA730,2201) mimics the K72R mutant protein in that it has no ATPase activity. The double mutant protein will form long extended filaments on ssDNA and facilitate LexA cleavage almost as well as wild-type, and do so in the presence of ATP. Unlike recA K72R, the recA E38K/ K72R double mutant promotes SOS induction in vivo after UV treatment. Thus, SOS induction does not require ATP hydrolysis by the RecA protein, but does require formation of extended RecA filaments. The RecA E38K/K72R protein represents an improved reagent for studies of the function of ATP hydrolysis by RecA in vivo and in vitro.

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“…Because the ssDNA-cellulose step purifies on the basis of ssDNA affinity, the specific activity of Rad59 is ϳ2-fold greater than the previous preparation. Escherichia coli RecA was purified as described (67) and was provided by Dr. Roberto Galletto in our laboratory. E. coli SSB protein was purified as described (68).…”

Section: Methodsmentioning

confidence: 99%

Rad51 Protein Controls Rad52-mediated DNA Annealing

Kantake

Sugiyama

et al. 2008

Journal of Biological Chemistry

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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 annealing, synthesis-dependent strand annealing, and crossover formation. Here we report that the S. cerevisiae DNA strand exchange protein, Rad51, prevents Rad52-mediated annealing of complementary ssDNA. Efficient inhibition is ATP-dependent and involves a specific interaction between Rad51 and Rad52. Free Rad51 can limit DNA annealing by Rad52, but the Rad51 nucleoprotein filament is even more effective. We also discovered that the budding yeast Rad52 paralog, Rad59 protein, partially restores Rad52-dependent DNA annealing in the presence of Rad51, suggesting that Rad52 and Rad59 function coordinately to enhance recombinational DNA repair either by directing the processed DSBs to repair by DNA strand annealing or by promoting second end capture to form a double Holliday junction. This regulation of Rad52-mediated annealing suggests a control function for Rad51 in deciding the recombination path taken for a processed DNA break; the ssDNA can be directed to either Rad51-mediated DNA strand invasion or to Rad52-mediated DNA annealing. This channeling determines the nature of the subsequent repair process and is consistent with the observed competition between these pathways in vivo.In Saccharomyces cerevisiae, the repair of DNA doublestranded breaks (DSBs) 3 is accomplished primarily by homologous recombination. Genes from the RAD52 epistasis group, including RAD50, RAD51, RAD52, RAD54, RAD55-57, RAD59, MRE11, XRS2, and RFA1, are responsible for this recombination-dependent DSB repair (1, 2). To repair a DSB, the DNA end is first processed to produce a 3Ј single-stranded tailed duplex DNA. The ssDNA is then channeled into one of the many recombinational pathways, which can be further categorized into RAD51-dependent and -independent pathways.RAD51-dependent recombination requires functions of RAD51, RAD52, RAD54, RAD55, RAD57, and RFA1 for efficient DNA repair (1). The central step of this pathway involves DNA strand invasion of homologous duplex DNA by the processed DSB complexed with Rad51 protein (3). DNA replication from the invading 3Ј-end replaces the genetic information missing from the broken dsDNA, and subsequent DNA pairing and resolution steps restore DNA integrity.RAD51-independent recombination requires RAD52 (4) and is enhanced by RAD59 (5-7). In addition to the recombination genes MRE11, RAD50, XRS2, RAD52, and RAD59 (6,8), this pathway also depends on MSH2, MSH3 (6, 9), RAD1, and RAD10 (10, 11). This RAD51-independent recombination is most easily assayed as DSB repair occurring between directly repeated sequences, by a mechanism termed single-stranded annealing (SSA) (8, 12). As implied, the central step of SSA is annealing between complementary singl...

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Biochemical Basis of the Constitutive Coprotease Activity of RecA P67W Protein

Cited by 8 publications

References 46 publications

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

Biochemical characterization of RecA variants that contribute to extreme resistance to ionizing radiation

RecA‐mediated SOS induction requires an extended filament conformation but no ATP hydrolysis

Rad51 Protein Controls Rad52-mediated DNA Annealing

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