Shyng‐Shiou F. Yuan

Mutations in BRCA1 and BRCA2 account for the majority of familial breast cancers. Cells with mutated BRCA1 or BRCA2 are hypersensitive to ionizing radiation (IR) and exhibit defective DNA repair. Both BRCA1 and BRCA2 have been reported to bind Rad51, a protein essential for homologous recombination and the recombinational repair of DNA double-strand breaks. In normal cells, a redistribution of Rad51 protein, manifested as formation of Rad51 nuclear foci, is seen upon IR treatment. Here we demonstrate that IR-induced Rad51 foci formation is aberrant in BRCA2- but not BRCA1-deficient tumor cells. In Capan-1 cells, which do not express functional BRCA2, there was little Rad51 foci formation in response to a wide range of radiation dosages. Moreover, forced expression of a fusion protein containing green fluorescent protein and the first Rad51-binding BRC repeat of BRCA2 in cells with wild-type BRCA2 rendered them hypersensitive to IR and cisplatin and compromised IR-induced Rad51 foci formation. In HCC1937 cells, which harbor mutation of BRCA1, IR-induced Rad51 foci were readily detected. This study suggests a requirement of BRCA2 protein for the IR-induced assembly of Rad51 complex in vivo.

Genetic studies in yeast have indicated a role of the RAD50 and MRE11 genes in homologous recombination, telomere length maintenance, and DNA repair processes. Here, we purify from nuclear extract of Raji cells a complex consisting of human Rad50, Mre11, and another protein factor with a size of about 95 kDa (p95), which is likely to be Nibrin, the protein encoded by the gene mutated in Nijmegen breakage syndrome. We show that the Rad50-Mre11-p95 complex possesses manganese-dependent single-stranded DNA endonuclease and 3′ to 5′ exonuclease activities. These nuclease activities are likely to be important for recombination, repair, and genomic stability. Genetic studies in yeast have indicated a role of the RAD50 and MRE11 genes in homologous recombination, telomere length maintenance, and DNA repair processes. Here, we purify from nuclear extract of Raji cells a complex consisting of human Rad50, Mre11, and another protein factor with a size of about 95 kDa (p95), which is likely to be Nibrin, the protein encoded by the gene mutated in Nijmegen breakage syndrome. We show that the Rad50-Mre11-p95 complex possesses manganese-dependent single-stranded DNA endonuclease and 3′ to 5′ exonuclease activities. These nuclease activities are likely to be important for recombination, repair, and genomic stability. Genetic studies on Saccharomyces cerevisiaemutants sensitive to ionizing radiation and to other agents that cause DNA double-stranded breaks have identified a large number of genetic loci required for the repair of such breaks. Many of these genes, including RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54, MRE11, and XRS2, show epistasis and are collectively known as the RAD52 epistasis group. Mutants of theRAD52 group also have defects of varying degrees in mitotic and meiotic recombination, which are initiated via DNA double-stranded break formation. Because meiotic recombination is essential for the proper segregation of homologous chromosomal pairs during meiosis I, the RAD52 group mutants often exhibit severe meiotic abnormalities, including inviability (see Refs. 1Klein H. Genetics. 1997; 147: 1533-1543Crossref PubMed Google Scholar and 2Shinohara M. Shita-Yamaguchi E. Buerstedde J.M. Shinagawa H. Ogawa H. Shinohara A. Genetics. 1997; 147: 1545-1556Crossref PubMed Google Scholarfor discussions and references).Extensive genetic evidence in yeast indicates that DNA double-stranded breaks are processed exonucleolytically, yielding 3′ overhanging single-stranded (ss) 1The abbreviations used are: sssingle-strandedGSTglutathione S-transferaseMOPS4-morpholinepropanesulfonic acid.1The abbreviations used are: sssingle-strandedGSTglutathione S-transferaseMOPS4-morpholinepropanesulfonic acid. tails of about 600 bases in length (3Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 4Sun H. Treco D. Szostak J.W. Cell. 1991; 64: 1155-1161Abstract Full Text PDF PubMed Scopus (422) Google Scholar). According to the double-stranded break repair model for recombination (5Szostak J.W. Orr-Weaver T.L. Rothstein R.J. Cell. 1993; 33: 25-35Abstract Full Text PDF Scopus (1745) Google Scholar), the 3′ ssDNA tails formed as a result of break processing are bound by recombination proteins, which then mediate a search for the chromosomal homolog and heteroduplex DNA formation with the homolog (5Szostak J.W. Orr-Weaver T.L. Rothstein R.J. Cell. 1993; 33: 25-35Abstract Full Text PDF Scopus (1745) Google Scholar). The RAD52 group genes may be divided into two categories. The first class consists of theRAD50, MRE11, and XRS2 genes, whose protein products are thought to be involved in the nucleolytic processing of DNA double-stranded breaks (6Shinohara A. Ogawa T. Trends Biochem. Sci. 1995; 20: 387-391Abstract Full Text PDF PubMed Scopus (3) Google Scholar). Consistent with this classification, the Rad50 and Mre11 proteins have been shown to be homologous to theEscherichia coli SbcC and SbcD proteins, which combine to form a complex with endonuclease and exonuclease activities (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google Scholar). The second category of the RAD52 group genes includesRAD51, RAD52, RAD54, RAD55, RAD57, and RDH54,whose products nucleate onto the ssDNA tails generated from break processing and then mediate the formation of heteroduplex DNA between the recombining chromosomes (1Klein H. Genetics. 1997; 147: 1533-1543Crossref PubMed Google Scholar, 2Shinohara M. Shita-Yamaguchi E. Buerstedde J.M. Shinagawa H. Ogawa H. Shinohara A. Genetics. 1997; 147: 1545-1556Crossref PubMed Google Scholar). Whether the Rad59 protein, which is homologous to Rad52 (8Bai Y. Symington L.S. Genes Dev. 1996; 10: 2025-2037Crossref PubMed Scopus (214) Google Scholar), also has a role in heteroduplex DNA formation remains to be established.Important insights concerning the mechanism by which theRAD52 group proteins form heteroduplex DNA have been garnered through biochemical studies of human and yeast proteins Sci. A. 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). as to the biochemical of the RAD50 and MRE11 encoded products is The Rad50 and Mre11 proteins are of genetic studies in yeast have indicated that are also for DNA Cell. PubMed Scopus Google and for the of telomere length PubMed Scopus Google Scholar). The human and MRE11 genes have been identified A. L. Cell. 1996; PubMed Scopus Google 1995; PubMed Scopus Google Scholar). Here, we purify a complex of human Rad50, Mre11, and a protein with size of 95 kDa from nuclear extract of Raji protein, is likely the protein to with Rad50 and Mre11 proteins from extract A. L. Cell. 1996; PubMed Scopus Google and identified to be Nibrin, the of the gene mutated in Nijmegen breakage H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. M. M. A. A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). biochemical studies that the Rad50-Mre11-p95 complex possesses endonuclease and a 3′ to 5′ exonuclease the Rad50 and Mre11 proteins in human in and proteins in and from E. shown in in the in nuclear extract of Raji cells a protein with size of about which in with the size of kDa for Rad50 protein 1995; PubMed Scopus Google Scholar). The a with size of about kDa in the Raji nuclear which in with the size of kDa for Mre11 protein A. L. Cell. 1996; PubMed Scopus Google human Rad50 and Mre11 proteins are in a complex A. L. Cell. 1996; PubMed Scopus Google Scholar). purify the a of nuclear by in of a second and and the of the complex from the by during the second on we a protein with the Rad50 and Mre11 the protein consisting of Rad50, Mre11, and in of with Rad50 and Mre11 proteins The protein used for the biochemical studies Rad50, Mre11, and in form The of this complex of Rad50, Mre11, and from of of Raji about of the Rad50-Mre11-p95 complex as the of and of this complex as the the used in the biochemical studies We that is to the protein with the size which in studies A. L. Cell. 1996; PubMed Scopus Google to be with Rad50 and Mre11 proteins in the to be in to Rad50 and Mre11 that is with a of the complex is of a in the protein Rad50 and Mre11 proteins are to the which possesses a ssDNA endonuclease (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google Scholar). this of to the Rad50-Mre11-p95 complex has a ssDNA endonuclease Rad50-Mre11-p95 complex with and to to the The in and with to the DNA shown in with the Rad50-Mre11-p95 complex the ssDNA into with that the Rad50-Mre11-p95 complex has ssDNA endonuclease complex has manganese-dependent endonuclease ssDNA with of Rad50-Mre11-p95 complex in of the of the with of and of and then in a the ssDNA in Rad50-Mre11-p95 the products formed as a result of nucleolytic DNA with of Rad50-Mre11-p95 complex in of the indicated of the with and then in a the DNA in Rad50-Mre11-p95 formed as a result of Rad50-Mre11-p95 nucleolytic the in and to to for a of the The the of ssDNA and the the of DNA nuclease is Rad50-Mre11-p95 and with of ssDNA in the of in the of and in the of for to the to the of the and the DNA in in the Rad50-Mre11-p95 nuclease also on double-stranded the nuclease the ssDNA with the double-stranded of the DNA by Rad50-Mre11-p95 a DNA shown in the Rad50-Mre11-p95 complex of the DNA into the the of the DNA a the of ssDNA the of about of the DNA been as with about of the ssDNA by of the of the nuclease the nuclease on which be by this the Rad50-Mre11-p95 nuclease the nuclease which is also on for (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google that the nuclease is to the Rad50-Mre11-p95 we from the of protein in to to of the Rad50-Mre11-p95 and the also used in nuclease with ssDNA as shown in the of nuclease the of Rad50-Mre11-p95 complex in the a used of as the we of the nuclease with the Rad50-Mre11-p95 complex with the Rad50-Mre11-p95 to to of the Rad50-Mre11-p95 complex and for nuclease ssDNA as of the nuclease with the Rad50-Mre11-p95 complex nuclease Rad50-Mre11-p95 complex in the and and for the Rad50-Mre11-p95 complex and for nuclease of the nuclease with the Rad50-Mre11-p95 complex nuclease Rad50-Mre11-p95 complex in the ssDNA in the products formed as a result of Rad50-Mre11-p95 nucleolytic the of the DNA generated as a result of Rad50-Mre11-p95 nucleolytic ssDNA with the Rad50-Mre11-p95 and the nucleolytic of the Rad50-Mre11-p95 DNA in and with to the of nucleolytic and other with and and with and with with that the of nucleolytic by the of a 3′ group. of the nucleolytic by by of the the of a 5′ the Rad50-Mre11-p95 nuclease 3′ and 5′ 3′ and 5′ DNA with Rad50-Mre11-p95 complex and DNA from the the nuclease complex in and with DNA with Rad50-Mre11-p95 and DNA from the with and by and then in The and to DNA with Rad50-Mre11-p95 and and DNA from the and with and and then as for The DNA in been with to the with the products formed as a result of Rad50-Mre11-p95 nucleolytic The are and Rad50-Mre11-p95 nuclease complex exonucleolytically, we DNA the 3′ 5′ with and then the DNA with Rad50-Mre11-p95 by of the in The and to and to of the shown in we that of the 3′ with the Rad50-Mre11-p95 complex for in of of the with the 5′ that the Rad50-Mre11-p95 complex also exonucleolytically, in the 3′ to 5′ The 3′ to 5′ exonuclease the endonuclease has a for complex has 3′ to 5′ exonuclease for exonuclease of the 5′ and 3′ DNA with of the Rad50-Mre11-p95 complex in of the a of the and with of The in a which with and and then and to and the in the 3′ DNA in and with of Rad50-Mre11-p95 protein complex for with with as have evidence that the Rad50-Mre11-p95 complex has endonuclease and 3′ to 5′ exonuclease activities. The Rad50-Mre11-p95 nuclease 3′ and 5′ that DNA in the nuclease complex DNA that are for DNA and for The nuclease in this protein complex likely in the Mre11 protein, the Mre11 protein has been to endonuclease the studies A. L. Cell. 1996; PubMed Scopus Google Scholar, H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), two protein with of and kDa to be with the Rad50-Mre11-p95 The identified to be the of the remains to be H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). These two protein are in of the Rad50-Mre11-p95 complex from with Nijmegen breakage which is by to ionizing radiation of a DNA repair to nuclear repair H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). may the nuclear of the is that the nuclease activities of the Rad50-Mre11-p95 complex and also have a role in the DNA repair to H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. M. M. A. A. Cell. Full Text Full Text PDF PubMed Scopus Google studies in yeast have the Rad50 and Mre11 proteins in homologous recombination (6Shinohara A. Ogawa T. Trends Biochem. Sci. 1995; 20: 387-391Abstract Full Text PDF PubMed Scopus (3) Google Scholar), repair (6Shinohara A. Ogawa T. Trends Biochem. Sci. 1995; 20: 387-391Abstract Full Text PDF PubMed Scopus (3) Google Scholar), repair by DNA Cell. PubMed Scopus Google Scholar), and in telomere length PubMed Scopus Google Scholar). the of of these proteins to in to DNA repair, the human Rad50-Mre11-p95 complex also important role in recombination and telomere length these chromosomal Rad50-Mre11-p95 nuclease activities may in with other protein such protein be a DNA which with the Rad50-Mre11-p95 endonuclease to a 3′ ssDNA (3Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 4Sun H. Treco D. Szostak J.W. Cell. 1991; 64: 1155-1161Abstract Full Text PDF PubMed Scopus (422) Google for the of and other proteins that in heteroduplex DNA formation Sci. A. 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). Genetic studies on Saccharomyces cerevisiaemutants sensitive to ionizing radiation and to other agents that cause DNA double-stranded breaks have identified a large number of genetic loci required for the repair of such breaks. Many of these genes, including RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54, MRE11, and XRS2, show epistasis and are collectively known as the RAD52 epistasis group. Mutants of theRAD52 group also have defects of varying degrees in mitotic and meiotic recombination, which are initiated via DNA double-stranded break formation. Because meiotic recombination is essential for the proper segregation of homologous chromosomal pairs during meiosis I, the RAD52 group mutants often exhibit severe meiotic abnormalities, including inviability (see Refs. 1Klein H. Genetics. 1997; 147: 1533-1543Crossref PubMed Google Scholar and 2Shinohara M. Shita-Yamaguchi E. Buerstedde J.M. Shinagawa H. Ogawa H. Shinohara A. Genetics. 1997; 147: 1545-1556Crossref PubMed Google Scholarfor discussions and genetic evidence in yeast indicates that DNA double-stranded breaks are processed exonucleolytically, yielding 3′ overhanging single-stranded (ss) 1The abbreviations used are: sssingle-strandedGSTglutathione S-transferaseMOPS4-morpholinepropanesulfonic acid.1The abbreviations used are: sssingle-strandedGSTglutathione S-transferaseMOPS4-morpholinepropanesulfonic acid. tails of about 600 bases in length (3Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 4Sun H. Treco D. Szostak J.W. Cell. 1991; 64: 1155-1161Abstract Full Text PDF PubMed Scopus (422) Google Scholar). According to the double-stranded break repair model for recombination (5Szostak J.W. Orr-Weaver T.L. Rothstein R.J. Cell. 1993; 33: 25-35Abstract Full Text PDF Scopus (1745) Google Scholar), the 3′ ssDNA tails formed as a result of break processing are bound by recombination proteins, which then mediate a search for the chromosomal homolog and heteroduplex DNA formation with the homolog (5Szostak J.W. Orr-Weaver T.L. Rothstein R.J. Cell. 1993; 33: 25-35Abstract Full Text PDF Scopus (1745) Google Scholar). The RAD52 group genes may be divided into two categories. The first class consists of theRAD50, MRE11, and XRS2 genes, whose protein products are thought to be involved in the nucleolytic processing of DNA double-stranded breaks (6Shinohara A. Ogawa T. Trends Biochem. Sci. 1995; 20: 387-391Abstract Full Text PDF PubMed Scopus (3) Google Scholar). Consistent with this classification, the Rad50 and Mre11 proteins have been shown to be homologous to theEscherichia coli SbcC and SbcD proteins, which combine to form a complex with endonuclease and exonuclease activities (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google Scholar). The second category of the RAD52 group genes includesRAD51, RAD52, RAD54, RAD55, RAD57, and RDH54,whose products nucleate onto the ssDNA tails generated from break processing and then mediate the formation of heteroduplex DNA between the recombining chromosomes (1Klein H. Genetics. 1997; 147: 1533-1543Crossref PubMed Google Scholar, 2Shinohara M. Shita-Yamaguchi E. Buerstedde J.M. Shinagawa H. Ogawa H. Shinohara A. Genetics. 1997; 147: 1545-1556Crossref PubMed Google Scholar). Whether the Rad59 protein, which is homologous to Rad52 (8Bai Y. Symington L.S. Genes Dev. 1996; 10: 2025-2037Crossref PubMed Scopus (214) Google Scholar), also has a role in heteroduplex DNA formation remains to be single-stranded acid. single-stranded acid. insights concerning the mechanism by which theRAD52 group proteins form heteroduplex DNA have been garnered through biochemical studies of human and yeast proteins Sci. A. 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). as to the biochemical of the RAD50 and MRE11 encoded products is The Rad50 and Mre11 proteins are of genetic studies in yeast have indicated that are also for DNA Cell. PubMed Scopus Google and for the of telomere length PubMed Scopus Google Scholar). The human and MRE11 genes have been identified A. L. Cell. 1996; PubMed Scopus Google 1995; PubMed Scopus Google Scholar). Here, we purify a complex of human Rad50, Mre11, and a protein with size of 95 kDa from nuclear extract of Raji protein, is likely the protein to with Rad50 and Mre11 proteins from extract A. L. Cell. 1996; PubMed Scopus Google and identified to be Nibrin, the of the gene mutated in Nijmegen breakage H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. M. M. A. A. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). biochemical studies that the Rad50-Mre11-p95 complex possesses endonuclease and a 3′ to 5′ exonuclease the Rad50 and Mre11 proteins in human in and proteins in and from E. shown in in the in nuclear extract of Raji cells a protein with size of about which in with the size of kDa for Rad50 protein 1995; PubMed Scopus Google Scholar). The a with size of about kDa in the Raji nuclear which in with the size of kDa for Mre11 protein A. L. Cell. 1996; PubMed Scopus Google human Rad50 and Mre11 proteins are in a complex A. L. Cell. 1996; PubMed Scopus Google Scholar). purify the a of nuclear by in of a second and and the of the complex from the by during the second on we a protein with the Rad50 and Mre11 the protein consisting of Rad50, Mre11, and in of with Rad50 and Mre11 proteins The protein used for the biochemical studies Rad50, Mre11, and in form The of this complex of Rad50, Mre11, and from of of Raji about of the Rad50-Mre11-p95 complex as the of and of this complex as the the used in the biochemical studies We that is to the protein with the size which in studies A. L. Cell. 1996; PubMed Scopus Google to be with Rad50 and Mre11 proteins in the to be in to Rad50 and Mre11 that is with a of the complex is of a in the protein Rad50 and Mre11 proteins are to the which possesses a ssDNA endonuclease (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google Scholar). this of to the Rad50-Mre11-p95 complex has a ssDNA endonuclease Rad50-Mre11-p95 complex with and to to the The in and with to the DNA shown in with the Rad50-Mre11-p95 complex the ssDNA into with that the Rad50-Mre11-p95 complex has ssDNA endonuclease the Rad50-Mre11-p95 nuclease also on double-stranded the nuclease the ssDNA with the double-stranded of the DNA by Rad50-Mre11-p95 a DNA shown in the Rad50-Mre11-p95 complex of the DNA into the the of the DNA a the of ssDNA the of about of the DNA been as with about of the ssDNA by of the of the nuclease the nuclease on which be by this the Rad50-Mre11-p95 nuclease the nuclease which is also on for (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google that the nuclease is to the Rad50-Mre11-p95 we from the of protein in to to of the Rad50-Mre11-p95 and the also used in nuclease with ssDNA as shown in the of nuclease the of Rad50-Mre11-p95 complex in the a used of as the we of the nuclease with the Rad50-Mre11-p95 complex with the Rad50-Mre11-p95 to to of the Rad50-Mre11-p95 complex and for nuclease ssDNA as of the nuclease with the Rad50-Mre11-p95 complex nuclease Rad50-Mre11-p95 complex in the and and for the Rad50-Mre11-p95 complex and for nuclease of the nuclease with the Rad50-Mre11-p95 complex nuclease Rad50-Mre11-p95 complex in the ssDNA in the products formed as a result of Rad50-Mre11-p95 nucleolytic the of the DNA generated as a result of Rad50-Mre11-p95 nucleolytic ssDNA with the Rad50-Mre11-p95 and the nucleolytic of the Rad50-Mre11-p95 DNA in and with to the of nucleolytic and other with and and with and with with that the of nucleolytic by the of a 3′ group. of the nucleolytic by by of the the of a 5′ the Rad50-Mre11-p95 nuclease 3′ and 5′ 3′ and 5′ DNA with Rad50-Mre11-p95 complex and DNA from the the nuclease complex in and with DNA with Rad50-Mre11-p95 and DNA from the with and by and then in The and to DNA with Rad50-Mre11-p95 and and DNA from the and with and and then as for The DNA in been with to the with the products formed as a result of Rad50-Mre11-p95 nucleolytic The are and Rad50-Mre11-p95 nuclease complex exonucleolytically, we DNA the 3′ 5′ with and then the DNA with Rad50-Mre11-p95 by of the in The and to and to of the shown in we that of the 3′ with the Rad50-Mre11-p95 complex for in of of the with the 5′ that the Rad50-Mre11-p95 complex also exonucleolytically, in the 3′ to 5′ The 3′ to 5′ exonuclease the endonuclease has a for complex has 3′ to 5′ exonuclease for exonuclease of the 5′ and 3′ DNA with of the Rad50-Mre11-p95 complex in of the a of the and with of The in a which with and and then and to and the in the 3′ DNA in and with of Rad50-Mre11-p95 protein complex for with with as have evidence that the Rad50-Mre11-p95 complex has endonuclease and 3′ to 5′ exonuclease activities. The Rad50-Mre11-p95 nuclease 3′ and 5′ that DNA in the nuclease complex DNA that are for DNA and for The nuclease in this protein complex likely in the Mre11 protein, the Mre11 protein has been to endonuclease the studies A. L. Cell. 1996; PubMed Scopus Google Scholar, H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), two protein with of and kDa to be with the Rad50-Mre11-p95 The identified to be the of the remains to be H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). These two protein are in of the Rad50-Mre11-p95 complex from with Nijmegen breakage which is by to ionizing radiation of a DNA repair to nuclear repair H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). may the nuclear of the is that the nuclease activities of the Rad50-Mre11-p95 complex and also have a role in the DNA repair to H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. M. M. A. A. Cell. Full Text Full Text PDF PubMed Scopus Google studies in yeast have the Rad50 and Mre11 proteins in homologous recombination (6Shinohara A. Ogawa T. Trends Biochem. Sci. 1995; 20: 387-391Abstract Full Text PDF PubMed Scopus (3) Google Scholar), repair (6Shinohara A. Ogawa T. Trends Biochem. Sci. 1995; 20: 387-391Abstract Full Text PDF PubMed Scopus (3) Google Scholar), repair by DNA Cell. PubMed Scopus Google Scholar), and in telomere length PubMed Scopus Google Scholar). the of of these proteins to in to DNA repair, the human Rad50-Mre11-p95 complex also important role in recombination and telomere length these chromosomal Rad50-Mre11-p95 nuclease activities may in with other protein such protein be a DNA which with the Rad50-Mre11-p95 endonuclease to a 3′ ssDNA (3Cao L. Alani E. Kleckner N. Cell. 1990; 61: 1089-1101Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 4Sun H. Treco D. Szostak J.W. Cell. 1991; 64: 1155-1161Abstract Full Text PDF PubMed Scopus (422) Google for the of and other proteins that in heteroduplex DNA formation Sci. A. 1997; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). the Rad50 and Mre11 proteins in human in and proteins in and from E. shown in in the in nuclear extract of Raji cells a protein with size of about which in with the size of kDa for Rad50 protein 1995; PubMed Scopus Google Scholar). The a with size of about kDa in the Raji nuclear which in with the size of kDa for Mre11 protein A. L. Cell. 1996; PubMed Scopus Google Scholar). The human Rad50 and Mre11 proteins are in a complex A. L. Cell. 1996; PubMed Scopus Google Scholar). purify the a of nuclear by in of a second and and the of the complex from the by during the second on we a protein with the Rad50 and Mre11 the protein consisting of Rad50, Mre11, and in of with Rad50 and Mre11 proteins The protein used for the biochemical studies Rad50, Mre11, and in form The of this complex of Rad50, Mre11, and from of of Raji about of the Rad50-Mre11-p95 complex as the of and of this complex as the the used in the biochemical studies We that is to the protein with the size which in studies A. L. Cell. 1996; PubMed Scopus Google to be with Rad50 and Mre11 proteins in the to be in to Rad50 and Mre11 that is with a of the complex is of a in the protein The Rad50 and Mre11 proteins are to the which possesses a ssDNA endonuclease (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google Scholar). this of to the Rad50-Mre11-p95 complex has a ssDNA endonuclease Rad50-Mre11-p95 complex with and to to the The in and with to the DNA shown in with the Rad50-Mre11-p95 complex the ssDNA into with that the Rad50-Mre11-p95 complex has ssDNA endonuclease the Rad50-Mre11-p95 nuclease also on double-stranded the nuclease the ssDNA with the double-stranded of the DNA by Rad50-Mre11-p95 a DNA shown in the Rad50-Mre11-p95 complex of the DNA into the the of the DNA a the of ssDNA the of about of the DNA been as with about of the ssDNA by The of the of the nuclease the nuclease on which be by this the Rad50-Mre11-p95 nuclease the nuclease which is also on for (7Connelly J.C. Leach D.R.F. Genes Cells. 1996; 1: 285-291Crossref PubMed Scopus (131) Google Scholar). that the nuclease is to the Rad50-Mre11-p95 we from the of protein in to to of the Rad50-Mre11-p95 and the also used in nuclease with ssDNA as shown in the of nuclease the of Rad50-Mre11-p95 complex in the a used of as the we of the nuclease with the Rad50-Mre11-p95 complex the of the DNA generated as a result of Rad50-Mre11-p95 nucleolytic ssDNA with the Rad50-Mre11-p95 and the nucleolytic of the Rad50-Mre11-p95 DNA in and with to the of nucleolytic and other with and and with and with with that the of nucleolytic by the of a 3′ group. of the nucleolytic by by of the the of a 5′ the Rad50-Mre11-p95 nuclease 3′ and 5′ Rad50-Mre11-p95 nuclease complex exonucleolytically, we DNA the 3′ 5′ with and then the DNA with Rad50-Mre11-p95 by of the in The and to and to of the shown in we that of the 3′ with the Rad50-Mre11-p95 complex for in of of the with the 5′ that the Rad50-Mre11-p95 complex also exonucleolytically, in the 3′ to 5′ The 3′ to 5′ exonuclease the endonuclease has a for We have evidence that the Rad50-Mre11-p95 complex has endonuclease and 3′ to 5′ exonuclease activities. The Rad50-Mre11-p95 nuclease 3′ and 5′ that DNA in the nuclease complex DNA that are for DNA and for The nuclease in this protein complex likely in the Mre11 protein, the Mre11 protein has been to endonuclease the studies A. L. Cell. 1996; PubMed Scopus Google Scholar, H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar), two protein with of and kDa to be with the Rad50-Mre11-p95 The identified to be the of the remains to be H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). These two protein are in of the Rad50-Mre11-p95 complex from with Nijmegen breakage which is by to ionizing radiation of a DNA repair to nuclear repair H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). may the nuclear of the is that the nuclease activities of the Rad50-Mre11-p95 complex and also have a role in the DNA repair to H. M. L. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, M. E. M. M. A. A. Cell. 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To assess the functions of the retinoblastoma protein (RB) during normal development, we have analyzed mouse embryos that lack a functional copy of the retinoblastoma gene (genotype: Rb-1 delta 20/Rb-1 delta 20). Our findings demonstrate that RB plays an important role in the regulation of the neuronal cell cycle. In mutant embryos, dividing cells are found well outside of the normal neurogenic regions in both the central and peripheral nervous systems. In addition to abnormal cell cycle regulation, however, the mutant embryos show two less expected phenotypes. First, many of the ectopically dividing cells die by apoptosis shortly after their entrance into S phase. In sensory ganglia, most nerve cells die by this process, beginning at about the same time as normal target-related neuronal death. Second, although the expression of certain differentiation markers such as N-CAM and Brn-3.0 appears to be near normal, nerve cells, especially in sensory ganglia, do not mature properly. Their morphology is stunted and expression of neuronal beta II tubulin is greatly reduced. Preferential reduction in the expression of TrkA, TrkB, and the low-affinity neurotrophin receptor p75LNGFR may be relevant to neuronal cell death and lack of neuronal differentiation seen in the mutant embryos. Primary cultures of dorsal root and trigeminal ganglion cells from later stage mutant embryos reveal a decrease in neuronal cell survival and in neurite outgrowth even in the presence of the appropriate neurotrophins. Taken together, these results suggest that the p110RB protein not only regulates progression through the cell cycle but is also important for cell survival and differentiation.