Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52.

Milne, G. Todd; Weaver, David T.

doi:10.1101/gad.7.9.1755

Cited by 221 publications

(211 citation statements)

References 27 publications

Supporting

Mentioning

195

Contrasting

Unclassified

Order By: Relevance

“…Rad52 binds to both single-and double-stranded DNA (7)(8)(9)(12)(13)(14)(15)(16), stimulates annealing of complementary DNA strands (7,(12)(13)(14)17), and promotes ligation of both cohesive and blunt-end fragments (9). Rad52 also interacts specifically with the Rad51 strand exchange enzyme (18,19), as well as the single-strand DNA-binding protein, RPA (20,21). Biochemical studies support the idea that Rad52 is a recombination mediator in that it optimizes catalysis of strand exchange by the Rad51 protein (22)(23)(24)(25)(26).…”

mentioning

confidence: 62%

Correlation of Biochemical Properties with the Oligomeric State of Human Rad52 Protein

Lloyd

Forget

Knight

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

The human Rad52 protein self-associates to form ringshaped oligomers, as well as higher order complexes of these rings. We have shown previously that there are two experimentally separable self-association domains in HsRad52, one in the N terminus (residues 1-192) responsible for assembly of individual subunits into rings, and one in the C terminus (residues 218 -418) responsible for higher order oligomerization of rings. Earlier studies suggest that the higher order complexes promote DNA end-joining, and others suggest that these complexes are relevant to in vivo Rad52 function. In this study we demonstrate that although inherent binding to single-stranded DNA depends on neither higher order complexes of Rad52 rings nor the ring-shaped oligomers themselves, higher order complexes are important for activities involving simultaneous interaction with more than one DNA molecule. This provides biochemical support for what may be an important in vivo function of Rad52.Chromosomal double strand breaks (DSBs) 1 occur both as a natural consequence of genome replication and division and through the damaging effects of ionizing radiation and environmental mutagens. Accurate repair of DSBs by homologous recombination is critical for the maintenance and propagation of genome integrity. In Saccharomyces cerevisiae members of the RAD52 epistasis group, RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, MRE11, and XRS2 were identified as components of the homologous recombination repair pathway because of the sensitivity of mutants to ionizing radiation (1). These gene products were also shown to play important roles in both mitotic and meiotic recombination (2). Mammalian counterparts for many of these RAD52 group genes have been discovered, and although there is substantial similarity between yeast and mammalian recombination pathways, both cellular and biochemical studies have revealed significant differences in the identity, regulation, and function of many of the component proteins (reviewed in Refs. 3-6).The human Rad52 protein (HsRad52) shares many similarities with yeast Rad52 regarding both its structure and function in homologous recombination. In either the absence or presence of DNA, Rad52 forms ring-shaped oligomers, ϳ10 nm in diameter, as well as higher order complexes of these rings (7-11). Rad52 binds to both single-and double-stranded DNA (7-9, 12-16), stimulates annealing of complementary DNA strands (7,(12)(13)(14)17), and promotes ligation of both cohesive and blunt-end fragments (9). Rad52 also interacts specifically with the Rad51 strand exchange enzyme (18, 19), as well as the single-strand DNA-binding protein, RPA (20,21). Biochemical studies support the idea that Rad52 is a recombination mediator in that it optimizes catalysis of strand exchange by the Rad51 protein (22-26). Park et al. (21) proposed a domain map for HsRad52 that included specific regions for self-association (residues 85-159), as well as interactions with the 34-kDa subunit of HsRPA (residues 221-280) and HsRad51 (residues 290 -330). This map ...

show abstract

mentioning

confidence: 62%

Correlation of Biochemical Properties with the Oligomeric State of Human Rad52 Protein

Lloyd

Forget

Knight

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The p181ANE vector (a generous gift from Wolfgang Frommer; unpublished results) contains a 2-pm origin of replication and the LEU2 selectable marker, and cloned inserts were placed under the control of the strong alcohol-dehydrogenase promoter ADHI for expression. The pDBL vector plasmid (2-pm origin, LEU2 marker; Milne and Weaver, 1993) and the pDBCl plasmid that contains the S. cerevisiue PTCl gene cloned into pDBL under the control of the ADHI promoter (Maeda et al, 1994) were obtained from Haruo Saito.…”

Section: Methodsmentioning

confidence: 99%

Protein Phosphatase Activity of Abscisic Acid Insensitive 1 (ABII) Protein from Arabidopsis Thaliana

Bertauche

Leung

Giraudat

1996

European Journal of Biochemistry

111

View full text Add to dashboard Cite

Mutations at the ABIl (abscisic acid insensitive 1) locus of the plant Arabidopsis thaliana cause a reduction in sensitivity to the plant hormone abscisic acid. The sequence of ABZl predicts a protein composed of an N-terminal domain that contains motifs for an EF-hand Caz+-binding site, and a Cterminal domain with similarities to protein serinekhreonine phosphatases 2C. We report here two sets of experimental evidence that indicate that ABIl has typical protein phosphatase 2C activity. First, expression of the ABIl C-terminal domain partially complemented the temperature-sensitive growth defect of a Saccharomyces cerevisiae protein phosphatase 2C mutant. Second, recombinant proteins that contained the ABIl C-terminal domain displayed in vitro phosphatase activity towards 'ZP-labelled casein, and this activity displayed Mg2+ or Mn'+ dependence and okadaic acid insensitivity typical of protein phosphatases 2C. Characterisation of recombinant proteins that contained various portions of ABIl indicated that the putative EF-hand motif is unlikely to mediate Caz+ regulation of the ABIl phosphatase activity at physiological CaZ+ concentrations, and may represent an EF-hand analogue rather than an EF-hand homologue. The abil-l mutation appeared to cause significant reduction in the phosphatase activity of ABII. These results are discussed in relation to the dominant phenotype of abil-1 over the wild-type allele in plants, and to the possible role of ABIl in abscisic acid signalling.

show abstract

“…Biochemical and genetic experiments provide support for this last possibility. Residual Rad51 activity is detected in the absence of mediators at high ssb concentrations (22,(49)(50)(51)(52), and overexpression of Rad51 can suppress the phenotypes of mutations that eliminate mediator function (66,69,93,94) Spontaneous Rad51 Foci. To determine whether Rad51 foci were present in undamaged yeast cells, aliquots from log phase cultures were examined without any prior damaging treatment.…”

Section: Reca-like Recombinases Assemble On Single-strand Dna (Ssdna)mentioning

confidence: 99%

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Gasior

Olivares

Ear

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

158

125

View full text Add to dashboard Cite

Members of the RecA family of recombinases from bacteriophage T4, Escherichia coli, yeast, and higher eukaryotes function in recombination as higher-order oligomers assembled on tracts of single-strand DNA (ssDNA). Biochemical studies have shown that assembly of recombinase involves accessory factors. These studies have identified a class of proteins, called recombination mediator proteins, that act by promoting assembly of recombinase on ssDNA tracts that are bound by ssDNA-binding protein (ssb). In the absence of mediators, ssb inhibits recombination reactions by competing with recombinase for DNA-binding sites. Here we briefly review mediated recombinase assembly and present results of new in vivo experiments. Immuno-double-staining experiments in Saccharomyces cerevisiae suggest that Rad51, the eukaryotic recombinase, can assemble at or near sites containing ssb (replication protein A, RPA) during the response to DNA damage, consistent with a need for mediator activity. Correspondingly, mediator gene mutants display defects in Rad51 assembly after DNA damage and during meiosis, although the requirements for assembly are distinct in the two cases. In meiosis, both Rad52 and Rad55͞57 are required, whereas either Rad52 or Rad55͞57 is sufficient to promote assembly of Rad51 in irradiated mitotic cells. Rad52 promotes normal amounts of Rad51 assembly in the absence of Rad55 at 30°C but not 20°C, accounting for the cold sensitivity of rad55 null mutants. Finally, we show that assembly of Rad51 is induced by radiation during S phase but not during G1, consistent with the role of Rad51 in repairing the spontaneous damage that occurs during DNA replication.H omologous recombination reactions promote repair of DNA ends formed by double-strand breaks (DSBs) and by replication fork collapse. Recombinational repair also allows cells to replicate past DNA lesions that block the progress of DNA polymerase. In phage T4, recombination is critical for initiating replication. In eukaryotes, homologous recombination is critical for accurate reductional segregation of chromosomes during meiosis. Finally, in prokaryotes, recombination allows horizontal transfer of alleles among and between bacteria and phage.At the center of homologous recombination are the recombinases, proteins that promote the formation of heteroduplex DNA. Of particular importance are the recombinases of the RecA family, including RecA in eubacteria, RadA in archea, Rad51 and Dmc1 in eukaryea, and the bacteriophage T4 UvsX protein.RecA-Like Recombinases Assemble on Single-Strand DNA (ssDNA).

show abstract

Dominant negative alleles of RAD52 reveal a DNA repair/recombination complex including Rad51 and Rad52.

Cited by 221 publications

References 27 publications

Correlation of Biochemical Properties with the Oligomeric State of Human Rad52 Protein

Correlation of Biochemical Properties with the Oligomeric State of Human Rad52 Protein

Protein Phosphatase Activity of Abscisic Acid Insensitive 1 (ABII) Protein from Arabidopsis Thaliana

Assembly of RecA-like recombinases: Distinct roles for mediator proteins in mitosis and meiosis

Contact Info

Product

Resources

About