The Experts below are selected from a list of 24933 Experts worldwide ranked by ideXlab platform
Stephen C. Kowalczykowski - One of the best experts on this subject based on the ideXlab platform.
-
Rad51 Protein Controls Rad52-mediated DNA Annealing
The Journal of biological chemistry, 2008Co-Authors: Noriko Kantake, Tomohiko Sugiyama, Stephen C. KowalczykowskiAbstract: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 cross-over 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.
-
The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 Protein
The Journal of biological chemistry, 2003Co-Authors: Noriko Kantake, Tomohiko Sugiyama, Richard D. Kolodner, Stephen C. KowalczykowskiAbstract:Replication Protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11)ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPAssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 ProteinssDNA nucleoProtein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 Protein more slowly than wild-type RPA and, as a consequence, Rad51 Protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 Protein can stimulate displacement of RPA(rfa1t11) from ssDNA by Rad51 Protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 Protein and that RPA displacement by Rad51 Protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.
-
Rad52 Protein associates with replication Protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement of RPA and presynaptic complex formation.
The Journal of biological chemistry, 2002Co-Authors: Tomohiko Sugiyama, Stephen C. KowalczykowskiAbstract: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 (ssDNA), but Rad52 Protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 Protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 Protein accelerates RPA displacement by Rad51 Protein. This acceleration probably involves direct interactions with both Rad51 Protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.
-
Tailed duplex DNA is the preferred substrate for Rad51 Protein-mediated homologous pairing
The EMBO journal, 2000Co-Authors: Alexander V. Mazin, Elena M. Zaitseva, Patrick Sung, Stephen C. KowalczykowskiAbstract:The repair of potentially lethal DNA double‐stranded breaks (DSBs) by homologous recombination requires processing of the broken DNA into a resected DNA duplex with a protruding 3′‐single‐stranded DNA (ssDNA) tail. Accordingly, the canonical models for DSB repair require invasion of an intact homologous DNA template by the 3′‐end of the ssDNA, a characteristic that the bacterial pairing Protein RecA possesses. Unexpectedly, we find that for the eukaryotic homolog, Rad51 Protein, the 5′‐end of ssDNA is more invasive than the 3′‐end. This pairing bias is unaffected by Rad52, Rad54 or Rad55–57 Proteins. However, further investigation reveals that, in contrast to RecA Protein, the preferred DNA substrate for Rad51 Protein is not ssDNA but rather dsDNA with ssDNA tails. This important distinction permits the Rad51 Proteins to promote DNA strand invasion using either 3′‐ or 5′‐ends with similar efficiency.
-
The DNA binding properties of Saccharomyces cerevisiae Rad51 Protein.
The Journal of biological chemistry, 1999Co-Authors: Elena M. Zaitseva, Eugene N. Zaitsev, Stephen C. KowalczykowskiAbstract:Saccharomyces cerevisiae Rad51 Protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange Proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 Protein, when compared with its prokaryotic homologue the Escherichia coli RecA Protein, we analyzed the DNA binding properties of the Rad51 Protein. Rad51 Protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 Protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of Protein. Without nucleotide cofactor, Rad51 Protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 Protein monomer per 6-9 nucleotides or base pairs. Therefore, Rad51 Protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 Protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 Protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoProtein filament; during synapsis, Rad51 Protein inactivates the homologous dsDNA partner by directly binding to it.
Wolf Dietrich Heyer - One of the best experts on this subject based on the ideXlab platform.
-
Rad54 Protein stimulates the Rad51 Protein-mediated DNI
2016Co-Authors: Jachen A. Solinger, Wolf Dietrich HeyerAbstract:Rad54 and Rad51 are important Proteins for the repair of doubleoc stranded DNA breaks by homologous recombination in euba karyotes. As previously shown, Rad51 Protein forms nucleoProtein dL filaments on single-stranded DNA, and Rad54 Protein directly pr interacts with such filaments to enhance synapsis, the homologous 13 pairing with a double-stranded DNA partner. Here we demonstrate In that Saccharomyces cerevisiae Rad54 Protein has an additional role al in the postsynaptic phase of DNA strand exchange by stimulating a heteroduplex DNA extension of established joint molecules in pr Rad51/Rpa-mediated DNA strand exchange. This function debe pended on the ATPase activity of Rad54 Protein and on specific Protein:Protein interactions between the yeast Rad54 and Rad51 ha Proteins. re
-
RAD54 controls access to the invading 3′-OH end after Rad51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae
Nucleic acids research, 2008Co-Authors: Wolf Dietrich HeyerAbstract:Rad51 is a key Protein in homologous recombination performing homology search and DNA strand invasion. After DNA strand exchange Rad51 Protein is stuck on the double-stranded heteroduplex DNA product of DNA strand invasion. This is a problem, because DNA polymerase requires access to the invading 3'-OH end to initiate DNA synthesis. Here we show that, the Saccharomyces cerevisiae dsDNA motor Protein Rad54 solves this problem by dissociating yeast Rad51 Protein bound to the heteroduplex DNA after DNA strand invasion. The reaction required species-specific interaction between both Proteins and the ATPase activity of Rad54 Protein. This mechanism rationalizes the in vivo requirement of Rad54 Protein for the turnover of Rad51 foci and explains the observed dependence of the transition from homologous pairing to DNA synthesis on Rad54 Protein in vegetative and meiotic yeast cells.
-
Loop 2 in Saccharomyces cerevisiae Rad51 Protein regulates filament formation and ATPase activity
Nucleic acids research, 2008Co-Authors: Xiao Ping Zhang, Vitold E. Galkin, Edward H. Egelman, Wolf Dietrich HeyerAbstract:Previous studies showed that the K342E substitution in the Saccharomyces cerevisiae Rad51 Protein increases the interaction with Rad54 Protein in the two-hybrid system, leads to increased sensitivity to the alkylating agent MMS and hyper-recombination in an oligonucleotide-mediated gene targeting assay. K342 localizes in loop 2, a region of Rad51 whose function is not well understood. Here, we show that Rad51-K342E displays DNA-independent and DNA-dependent ATPase activities, owing to its ability to form filaments in the absence of a DNA lattice. These filaments exhibit a compressed pitch of 81 A, whereas filaments of wild-type Rad51 and Rad51-K342E on DNA form extended filaments with a 97 A pitch. Rad51-K342E shows near normal binding to ssDNA, but displays a defect in dsDNA binding, resulting in less stable Protein-dsDNA complexes. The mutant Protein is capable of catalyzing the DNA strand exchange reaction and is insensitive to inhibition by the early addition of dsDNA. Wild-type Rad51 Protein is inhibited under such conditions, because of its ability to bind dsDNA. No significant changes in the interaction between Rad51-K342E and Rad54 could be identified. These findings suggest that loop 2 contributes to the primary DNA-binding site in Rad51, controlling filament formation and ATPase activity.
-
rad54 a swi2 snf2 like recombinational repair Protein disassembles Rad51 dsdna filaments
Molecular Cell, 2002Co-Authors: Jachen A. Solinger, Konstantin Kiianitsa, Wolf Dietrich HeyerAbstract:Rad54 Protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel Protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Here we show that Rad54 Protein dissociates Rad51 from nucleoProtein filaments formed on dsDNA. Addition of Rad54 Protein overcomes inhibition of DNA strand exchange by Rad51 Protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underlines the importance of specific Rad54-Rad51 Protein interactions. Rad51 Protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. We suggest that Rad54 Protein is involved in the turnover of Rad51-dsDNA filaments.
-
Rad54 Protein Exerts Diverse Modes of ATPase Activity on Duplex DNA Partially and Fully Covered with Rad51 Protein
The Journal of biological chemistry, 2002Co-Authors: Konstantin Kiianitsa, Jachen A. Solinger, Wolf Dietrich HeyerAbstract:Rad54 Protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. Herein, we address the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. We show that Saccharomyces cerevisiae Rad54 exerts distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 Protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with Protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 Protein but to a lesser extent. In contrast, the interaction of Rad54 Protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 Protein hydrolyzes ATP in a reduced mode at 60-80% of its rate on Protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. We suggest that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination.
Stephen C West - One of the best experts on this subject based on the ideXlab platform.
-
Conformational changes modulate the activity of human Rad51 Protein.
Journal of molecular biology, 2004Co-Authors: Yilun Liu, Andrzej Stasiak, Alicja Z. Stasiak, Jean-yves Masson, Michael J. Mcilwraith, Stephen C WestAbstract:Abstract Homologous recombination provides a major pathway for the repair of DNA double-strand breaks in mammalian cells. Defects in homologous recombination can lead to high levels of chromosomal translocations or deletions, which may promote cell transformation and cancer development. A key component of this process is Rad51. In comparison to RecA, the bacterial homologue, human Rad51 Protein exhibits low-level strand-exchange activity in vitro. This activity can, however, be stimulated by the presence of high salt. Here, we have investigated the mechanistic basis for this stimulation. We show that high ionic strength favours the co-aggregation of Rad51–single-stranded DNA (ssDNA) nucleoProtein filaments with naked duplex DNA, to form a complex in which the search for homologous sequences takes place. High ionic strength allows differential binding of Rad51 to ssDNA and double-stranded DNA (dsDNA), such that ssDNA–Rad51 interactions are unaffected, whereas those between Rad51 and dsDNA are destabilised. Most importantly, high salt induces a conformational change in Rad51, leading to the formation of extended nucleoProtein filaments on ssDNA. These extended filaments mimic the active form of the Escherichia coli RecA–ssDNA filament that exhibits efficient strand-exchange activity.
-
heteroduplex formation by human Rad51 Protein effects of dna end structure hrp a and hrad52
Journal of Molecular Biology, 1999Co-Authors: Peter Baumann, Stephen C WestAbstract:Abstract Purified human Rad51 Protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 Protein. Joint molecule formation required the presence of a 3′ or 5′-overhang on the duplex DNA substrate and initiated preferentially at the 5′-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3′-5′ direction relative to the single-stranded DNA. The human single-strand DNA-binding Protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding Protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding Protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.
-
role of the human Rad51 Protein in homologous recombination and double stranded break repair
Trends in Biochemical Sciences, 1998Co-Authors: Peter Baumann, Stephen C WestAbstract:Eukaryotic cells possess several mechanisms for repairing double-stranded breaks in DNA. One mechanism involves genetic recombination with an intact sister duplex. The recent identification of the Rad51 Protein, a eukaryotic homologue of Escherichia coli RecA, represents a landmark discovery in our understanding of the key reactions in this repair pathway. Rad51 is similar to RecA, both biochemically and structurally: it promotes homologous pairing and strand exchange within a regular nucleoProtein filament. The isolation of yeast and human RecA homologues shows that homologous recombination and recombinational repair have been conserved throughout evolution. The goal is now to identify other factors involved in recombinational repair and to define their roles in this essential process.
-
The human Rad51 Protein: polarity of strand transfer and stimulation by hRP-A.
The EMBO journal, 1997Co-Authors: Peter Baumann, Stephen C WestAbstract:The human Rad51 Protein is homologous to the Escherichia coli RecA Protein and catalyses homologous pairing and strand transfer reactions in vitro . Using single‐stranded circular and homologous linear duplex DNA, we show that hRad51 forms stable joint molecules by transfer of the 5′ end of the complementary strand of the linear duplex to the ssDNA. The polarity of strand transfer is therefore 3′ to 5′, defined relative to the ssDNA on which hRad51 initiates filament formation. This polarity is opposite to that observed with RecA. Homologous pairing and strand transfer require stoichiometric amounts of hRad51, corresponding to one hRad51 monomer per three nucleotides of ssDNA. Joint molecules are not observed when the Protein is present in limiting or excessive amounts. The human ssDNA binding‐Protein, hRP‐A, stimulates hRad51‐mediated reactions. Its effect is consistent with a role in the removal of secondary structures from ssDNA, thereby facilitating the formation of continuous Rad51 filaments.
-
Distribution of the Rad51 recombinase in human and mouse spermatocytes
The EMBO journal, 1997Co-Authors: Andrew L. Barlow, Stephen C West, Fiona E. Benson, HulténAbstract:In vitro, the human Rad51 Protein (hRad51) promotes homologous pairing and strand exchange reactions suggestive of a key role in genetic recombination. To analyse its role in this process, polyclonal antibodies raised against hRad51 were used to study the distribution of Rad51 in human and mouse spermatocytes during meiosis I. In human spermatocytes, hRad51 was found to form discrete nuclear foci from early zygotene to late pachytene. The foci always co-localized with lateral element Proteins, components of the synaptonemal complex (SC). During zygotene, the largest foci were present in regions undergoing synapsis, suggesting that Rad51 is a component of early recombination nodules. Pachytene nuclei showed a greatly reduced level of Rad51 labelling, with the exceptions of any asynapsed autosomes and XY segments, which were intensely labelled. The distribution of Rad51 in mouse spermatocytes was similar to that found in human spermatocytes, except that in this case Rad51 was detectable at leptotene. From these results, we conclude that the Rad51 Protein has a role in the interhomologue interactions that occur during meiotic recombination. These interactions are spatially and temporally associated with synapsis during meiotic prophase I.
Jachen A. Solinger - One of the best experts on this subject based on the ideXlab platform.
-
Rad54 Protein stimulates the Rad51 Protein-mediated DNI
2016Co-Authors: Jachen A. Solinger, Wolf Dietrich HeyerAbstract:Rad54 and Rad51 are important Proteins for the repair of doubleoc stranded DNA breaks by homologous recombination in euba karyotes. As previously shown, Rad51 Protein forms nucleoProtein dL filaments on single-stranded DNA, and Rad54 Protein directly pr interacts with such filaments to enhance synapsis, the homologous 13 pairing with a double-stranded DNA partner. Here we demonstrate In that Saccharomyces cerevisiae Rad54 Protein has an additional role al in the postsynaptic phase of DNA strand exchange by stimulating a heteroduplex DNA extension of established joint molecules in pr Rad51/Rpa-mediated DNA strand exchange. This function debe pended on the ATPase activity of Rad54 Protein and on specific Protein:Protein interactions between the yeast Rad54 and Rad51 ha Proteins. re
-
rad54 a swi2 snf2 like recombinational repair Protein disassembles Rad51 dsdna filaments
Molecular Cell, 2002Co-Authors: Jachen A. Solinger, Konstantin Kiianitsa, Wolf Dietrich HeyerAbstract:Rad54 Protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel Protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Here we show that Rad54 Protein dissociates Rad51 from nucleoProtein filaments formed on dsDNA. Addition of Rad54 Protein overcomes inhibition of DNA strand exchange by Rad51 Protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underlines the importance of specific Rad54-Rad51 Protein interactions. Rad51 Protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. We suggest that Rad54 Protein is involved in the turnover of Rad51-dsDNA filaments.
-
Rad54 Protein Exerts Diverse Modes of ATPase Activity on Duplex DNA Partially and Fully Covered with Rad51 Protein
The Journal of biological chemistry, 2002Co-Authors: Konstantin Kiianitsa, Jachen A. Solinger, Wolf Dietrich HeyerAbstract:Rad54 Protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. Herein, we address the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. We show that Saccharomyces cerevisiae Rad54 exerts distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 Protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with Protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 Protein but to a lesser extent. In contrast, the interaction of Rad54 Protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 Protein hydrolyzes ATP in a reduced mode at 60-80% of its rate on Protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. We suggest that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination.
-
Rad54, a Swi2/Snf2-like Recombinational Repair Protein, Disassembles Rad51:dsDNA Filaments
Molecular cell, 2002Co-Authors: Jachen A. Solinger, Konstantin Kiianitsa, Wolf Dietrich HeyerAbstract:Rad54 Protein is a member of the Swi2/Snf2-like family of DNA-dependent/stimulated ATPases that dissociate and remodel Protein complexes on dsDNA. Rad54 functions in the recombinational DNA repair (RAD52) pathway. Here we show that Rad54 Protein dissociates Rad51 from nucleoProtein filaments formed on dsDNA. Addition of Rad54 Protein overcomes inhibition of DNA strand exchange by Rad51 Protein bound to substrate dsDNA. Species preference in the Rad51 dissociation and DNA strand exchange assays underlines the importance of specific Rad54-Rad51 Protein interactions. Rad51 Protein is unable to release dsDNA upon ATP hydrolysis, leaving it stuck on the heteroduplex DNA product after DNA strand exchange. We suggest that Rad54 Protein is involved in the turnover of Rad51-dsDNA filaments.
-
Rad54 Protein stimulates the postsynaptic phase of Rad51 Protein-mediated DNA strand exchange
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Jachen A. Solinger, Wolf Dietrich HeyerAbstract:Rad54 and Rad51 are important Proteins for the repair of double-stranded DNA breaks by homologous recombination in eukaryotes. As previously shown, Rad51 Protein forms nucleoProtein filaments on single-stranded DNA, and Rad54 Protein directly interacts with such filaments to enhance synapsis, the homologous pairing with a double-stranded DNA partner. Here we demonstrate that Saccharomyces cerevisiae Rad54 Protein has an additional role in the postsynaptic phase of DNA strand exchange by stimulating heteroduplex DNA extension of established joint molecules in Rad51/Rpa-mediated DNA strand exchange. This function depended on the ATPase activity of Rad54 Protein and on specific Protein:Protein interactions between the yeast Rad54 and Rad51 Proteins.
Tomohiko Sugiyama - One of the best experts on this subject based on the ideXlab platform.
-
Rad51 Protein Controls Rad52-mediated DNA Annealing
The Journal of biological chemistry, 2008Co-Authors: Noriko Kantake, Tomohiko Sugiyama, Stephen C. KowalczykowskiAbstract: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 cross-over 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.
-
The recombination-deficient mutant RPA (rfa1-t11) is displaced slowly from single-stranded DNA by Rad51 Protein
The Journal of biological chemistry, 2003Co-Authors: Noriko Kantake, Tomohiko Sugiyama, Richard D. Kolodner, Stephen C. KowalczykowskiAbstract:Replication Protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11)ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPAssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 ProteinssDNA nucleoProtein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 Protein more slowly than wild-type RPA and, as a consequence, Rad51 Protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 Protein can stimulate displacement of RPA(rfa1t11) from ssDNA by Rad51 Protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 Protein and that RPA displacement by Rad51 Protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.
-
Rad52 Protein associates with replication Protein A (RPA)-single-stranded DNA to accelerate Rad51-mediated displacement of RPA and presynaptic complex formation.
The Journal of biological chemistry, 2002Co-Authors: Tomohiko Sugiyama, Stephen C. KowalczykowskiAbstract: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 (ssDNA), but Rad52 Protein alleviates this inhibition. Here we show that Rad51 filament formation is simultaneous with displacement of RPA from ssDNA. This displacement is initiated by a rate-limiting nucleation of Rad51 Protein onto ssDNA complex, followed by rapid elongation of the filament. Rad52 Protein accelerates RPA displacement by Rad51 Protein. This acceleration probably involves direct interactions with both Rad51 Protein and RPA. Detection of a Rad52-RPA-ssDNA co-complex suggests that this co-complex is an intermediate in the displacement process.
-
rad52 Protein stimulates dna strand exchange by Rad51 and replication Protein a
Nature, 1998Co-Authors: Tomohiko Sugiyama, Elena Zaitseva, Stephen C. KowalczykowskiAbstract:The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability.The repair of such lesions is strongly dependent on Proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2)1,2, as well as the RFA13,4 and RAD59 genes5. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair2, but almost nothing is known about the biochemical role of Rad52 Protein. Rad51 Protein promotes DNA strand exchange6,7,8 and acts similarly to RecA Protein9. Yeast Rad52 Protein interacts with Rad51 Protein10,11, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA12. We find that Rad52 Protein stimulates DNA strand exchange by targeting Rad51 Protein to a complex of replication Protein A (RPA) with single-stranded DNA. Rad52 Protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 Protein and RPA, implying that specific Protein–Protein interactions between Rad52 Protein, Rad51 Protein and RPA are required.
-
a single stranded dna binding Protein is needed for efficient presynaptic complex formation by the saccharomyces cerevisiae Rad51 Protein
Journal of Biological Chemistry, 1997Co-Authors: Tomohiko Sugiyama, Elena Zaitseva, Stephen C. KowalczykowskiAbstract:Protein-promoted DNA strand exchange requires formation of an active presynaptic complex between the DNA-pairing Protein and single-stranded DNA (ssDNA). Formation of such a contiguous filament is stimulated by a ssDNA-binding Protein. Here, the effects of replication Protein A (RPA) on presynaptic complex formation and DNA strand exchange activities of Rad51 Protein were examined. Presynaptic complex formation was assessed by measuring ATP hydrolysis. With phiX174 ssDNA, the ATPase activity of Rad51 Protein is stimulated approximately 1.4-fold by RPA, provided that Rad51 Protein is in excess of the ssDNA concentration; otherwise, RPA inhibits ATPase activity. In contrast, with ssDNA devoid of secondary structure (poly(dT), poly(dA), poly(dI), and etheno-M13 DNA), RPA does not stimulate the already elevated ATPase activity of Rad51 Protein, but inhibits activity at low Rad51 Protein concentrations. These results suggest that Rad51 Protein and RPA exclude one another from ssDNA by competing for the same binding sites and that RPA exerts its effect on presynaptic complex formation by eliminating secondary structure to which Rad51 Protein is bound nonproductively. DNA strand exchange catalyzed by Rad51 Protein is also greatly stimulated by RPA. The optimal stoichiometry for stimulation is approximately 20-30 nucleotides of ssDNA/RPA heterotrimer. The ssDNA-binding Protein of Escherichia coli can substitute for RPA, showing that the role of RPA is not specific. We conclude that RPA affects both presynaptic complex formation and DNA strand exchange via changes in DNA structure, employing the same mechanism used by the ssDNA-binding Protein to effect change in E. coli RecA Protein activity.
