Homologous Pairing Promoted by the Human Rad52 Protein

Kagawa, Wataru; Kurumizaka, Hitoshi; Ikawa, Shiro; Yokoyama, Shigeyuki; Shibata, Takehiko

doi:10.1074/jbc.m104938200

Cited by 143 publications

(169 citation statements)

References 64 publications

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“…human RAD52 proteins (29,35). When mTBPIP/HOP2 was incubated with dsDNA before DMC1 and ssDNA were added, the D-loop yield at 10 min was increased about 2.9-fold relative to that promoted by DMC1 alone, but the D-loop dissociation was still observed (Fig.…”

Section: Tbpip/hop2 Enhances the Dmc1-mediated Homologous Pairingmentioning

confidence: 99%

“…Overexpression and Purification of the Human DMC1 and RAD51 Proteins-The human RAD51 protein was purified as described previously (29). The human DMC1 gene was inserted into the pET-15b plasmid (Novagen) at the NdeI-BamHI sites, and the protein was overexpressed in the E. coli strain BL21-CodonPlus(DE3)-RIL (Stratagene) as an N-terminally His 6 -tagged protein (30).…”

Section: Overexpression and Purification Of The Mouse And Human Tbpipmentioning

confidence: 99%

“…Instead the cells were gently lysed using sarkosyl as described previously (29). The pGsat4 DNA was created by inserting a 198-base pair fragment of the human ␣-satellite sequence into the pGEM-T Easy vector (Promega).…”

Section: Overexpression and Purification Of The Mouse And Human Tbpipmentioning

confidence: 99%

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Positive Role of the Mammalian TBPIP/HOP2 Protein in DMC1-mediated Homologous Pairing

Enomoto¹,

Kinebuchi²,

Sato

et al. 2004

Journal of Biological Chemistry

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In meiosis, homologous recombination preferentially occurs between homologous chromosomes rather than between sister chromatids, which is opposite to the bias of mitotic recombinational repair. The TBPIP/HOP2 protein is a factor that ensures the proper pairing of homologous chromosomes during meiosis. In the present study, we found that the purified mouse TBPIP/ HOP2 protein stimulated homologous pairing catalyzed by the meiotic DMC1 recombinase in vitro. In contrast, TBPIP/HOP2 did not stimulate homologous pairing by RAD51, which is another homologous pairing protein acting in both meiotic and mitotic recombination. The positive effect of TBPIP/HOP2 in the DMC1-mediated homologous pairing was only observed when TBPIP/ HOP2 first binds to double-stranded DNA, not to singlestranded DNA, before the initiation of the homologous pairing reaction. Deletion analyses revealed that the C-terminal basic region of TBPIP/HOP2 is required for efficient DNA binding and is also essential for its homologous pairing stimulation activity. Therefore, these results suggest that TBPIP/HOP2 directly binds to DNA and functions as an activator for DMC1 during the homologous pairing step in meiosis.During meiosis, two successive rounds of nuclear division, meiosis I and meiosis II, are promoted with a single round of DNA replication. As a result, diploid cells produce haploid gametes in eukaryotes. In the cell division at meiosis I, homologous chromosomes are aligned, and a high level of recombination occurs between homologous chromosomes but not between sister chromatids. This preferential recombination between homologous chromosomes, called homologous recombination, ensures their correct segregation at meiosis I through the formation of chiasmata, which physically connect homologous chromosomes.Homologous recombination is initiated by double strand break (DSB) 1 formation by SPO11 at the initiation sites for recombination (1-3). Then a single-stranded DNA (ssDNA) tail derived from a DSB site invades the homologous doublestranded DNA (dsDNA) to form a heteroduplex. This strand invasion step, called homologous pairing, is the key step in homologous recombination. In Escherichia coli, the RecA protein catalyzes the homologous pairing step (4, 5). Two eukaryotic homologues of RecA, the RAD51 and DMC1 proteins, which are conserved from yeast to human, have been identified (6 -9) and have been shown to catalyze homologous pairing in vitro (10 -15). The RAD51 gene is expressed in both mitotic and meiotic cells, but the DMC1 gene functions only in meiotic cells. Disruption of the Rad51 gene results in early embryonic lethality in mice (16,17). In chicken DT40 cells, the RAD51 gene disruption causes the accumulation of spontaneous chromosomal breaks and significantly reduces the recombination frequency between sister chromatids (18,19). In contrast, disruption of the Dmc1 gene does not cause lethality in mice, but the dmc1 knock-out mice are sterile with an arrest of gametogenesis in the first meiotic prophase (20,21). Therefore, DMC1 is a me...

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Section: Tbpip/hop2 Enhances the Dmc1-mediated Homologous Pairingmentioning

confidence: 99%

Section: Overexpression and Purification Of The Mouse And Human Tbpipmentioning

confidence: 99%

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Positive Role of the Mammalian TBPIP/HOP2 Protein in DMC1-mediated Homologous Pairing

Enomoto¹,

Kinebuchi²,

Sato

et al. 2004

Journal of Biological Chemistry

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“…The immunoprecipitates were subjected to SDS-PAGE followed by Western blot analysis. To obtain a rabbit antibody to RAD51, the human Rad51 protein was expressed as a recombinant protein in the Escherichia coli strain JM109 (DE3) (Kagawa et al, 2001), and was purified as described previously (Kurumizaka et al, 1999). Detection of target proteins was with an enhanced chemiluminescence detection system (Amersham Biosciences).…”

Section: Protein Analysesmentioning

confidence: 99%

HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination

et al. 2006

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“…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)(8)(9)(10)(11). 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).…”

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confidence: 99%

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

Lloyd

Forget

Knight

2002

Journal of Biological Chemistry

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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 ...

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Homologous Pairing Promoted by the Human Rad52 Protein

Cited by 143 publications

References 64 publications

Positive Role of the Mammalian TBPIP/HOP2 Protein in DMC1-mediated Homologous Pairing

Positive Role of the Mammalian TBPIP/HOP2 Protein in DMC1-mediated Homologous Pairing

HIV-1 Vpr induces ATM-dependent cellular signal with enhanced homologous recombination

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

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