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• W2022066102 abstract "A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1–/– cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis. A variety of cellular proteins has the ability to recognize DNA lesions induced by the anti-cancer drug cisplatin, with diverse consequences on their repair and on the therapeutic effectiveness of this drug. We report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to RAD52, a protein involved in homologous recombination and DNA double-strand break repair. Ablation of RDM1 in the chicken B cell line DT40 led to a more than 3-fold increase in sensitivity to cisplatin. However, RDM1–/– cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate. The RDM1 protein displays a nucleic acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA as well as double-stranded DNA, on which it assembles filament-like structures. Notably, RDM1 recognizes DNA distortions induced by cisplatin-DNA adducts in vitro. Finally, human RDM1 transcripts are abundant in the testis, suggesting a possible role during spermatogenesis. The cytotoxic activity of cisplatin (cis-diamminedichloroplatinum (II)) is thought to be due to its interaction with purine bases of our chromosomes and the various DNA adducts that ensue, including monoadducts and interstrand cross-links (ICLs), 1The abbreviations used are: ICL, interstrand cross-links; NER, nucleotide excision repair; CS, Cockayne syndrome; dsDNA, double-stranded DNA; DSB, double-stranded break; GGR, global genome repair; HMG, high mobility group; HR, homologous recombination; MMR, mismatch repair; MMS, methylmethane sulfonate; ORF, open reading frame; RRM, RNA recognition motif; SSA, single-strand annealing; ssDNA, single-stranded DNA; TCR, transcription-coupled repair; XP, xeroderma pigmentosum; IPTG, isopropyl 1-thio-β-d-galactopyranoside; MES, 4-morpholineethanesulfonic acid; aa, amino acid. as well as the predominant (1,3- and 1,2-) intrastrand cross-links (1Wozniak K. Blasiak J. Acta Biochim. Pol. 2002; 49: 583-596Crossref PubMed Scopus (132) Google Scholar, 2Zamble D.B. Lippard S.J. Trends Biochem. Sci. 1995; 20: 435-439Abstract Full Text PDF PubMed Scopus (482) Google Scholar). Cisplatin-DNA adducts perturb replication and transcription and trigger sophisticated repair machineries. Cisplatin-DNA adducts are removed primarily by nucleotide excision repair (NER), which also operates in the repair of UV-induced lesions, DNA cross-links of various origins, and bulky DNA adducts (3Friedberg E.C. Nat. Rev. Cancer. 2001; 1: 22-33Crossref PubMed Scopus (592) Google Scholar, 4Wood R.D. Araujo S.J. Ariza R.R. Batty D.P. Biggerstaff M. Evans E. Gaillard P.H. Gunz D. Koberle B. Kuraoka I. Moggs J.G. Sandall J.K. Shivji M.K. Cold Spring Harbor Symp. Quant. Biol. 2000; 65: 173-182Crossref PubMed Scopus (48) Google Scholar). NER has been elucidated thanks in part to the use of cell lines derived from patients with xeroderma pigmentosum (XP) and Cockayne syndrome (CS), in which NER was found to be deficient (5Bootsma D. Kraemer K.H. Cleaver J.E. Hoeijmakers J.H. Vogelstein B. Kinzler K.W. The Genetic Basis of Human Cancer. McGraw-Hill Inc., New York1998: 245-274Google Scholar). NER has been subdivided in two subpathways that operate either on the transcribed strand of transcriptionally active genes (transcription-coupled repair (TCR)) or on the nontranscribed strand of active genes and in transcriptionally silent regions of the genome (global genome repair (GGR)) (6Hanawalt P.C. Oncogene. 2002; 21: 8949-8956Crossref PubMed Scopus (358) Google Scholar). The main difference between these pathways resides in the mechanisms by which they achieve specific recognition of a DNA lesion. In GGR, damage recognition involves the XPC-hHR23B complex, XPA, and RPA (7Reardon J.T. Sancar A. Genes Dev. 2003; 17: 2539-2551Crossref PubMed Scopus (148) Google Scholar, 8Thoma B.S. Vasquez K.M. Mol. Carcinog. 2003; 38: 1-13Crossref PubMed Scopus (107) Google Scholar), as well as the UV-damaged DNA binding complex (DDB) (9Fitch M.E. Nakajima S. Yasui A. Ford J.M. J. Biol. Chem. 2003; 278: 46906-46910Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In TCR, DNA damage is sensed by the RNA polymerase II, and repair is engaged by the transcription-coupled repair-specific factors CSA and CSB. Recognition of the lesion is followed by the sequential recruitment of proteins that mediate stages of repair common to GGR and TCR as follows: formation of an open, “pre-incision” complex, excision of a DNA fragment containing the lesion, and repair DNA synthesis (10de Laat W.L. Jaspers N.G. Hoeijmakers J.H. Genes Dev. 1999; 13: 768-785Crossref PubMed Scopus (922) Google Scholar). Although NER is the main pathway for the removal of intrastrand DNA cross-links, the molecular mechanisms that ensure repair of ICLs are still unclear. Current models suggest that such repair is initiated by DNA double-stranded breaks (DSBs) that are generated by the collapse of replication forks at the sites of lesion. These breaks are then acted upon by components of the NER and homologous recombination (HR) pathways, leading to the removal of the lesion (11Dronkert M.L. Kanaar R. Mutat. Res. 2001; 486: 217-247Crossref PubMed Scopus (487) Google Scholar). Biochemical evidence of DNA replication-mediated DSBs at sites of cross-links has been reported recently (12Bessho T. J. Biol. Chem. 2003; 278: 5250-5254Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Removal of cisplatin-DNA cross-links contributes to resistance of tumors to the drug, a phenomenon frequently manifested during chemotherapy (2Zamble D.B. Lippard S.J. Trends Biochem. Sci. 1995; 20: 435-439Abstract Full Text PDF PubMed Scopus (482) Google Scholar). Thus, increased expression of NER components and/or higher levels of cross-link excision activities have been detected in cancers that fail to respond to the drug (Ref. 13Selvakumaran M. Pisarcik D.A. Bao R. Yeung A.T. Hamilton T.C. Cancer Res. 2003; 63: 1311-1316PubMed Google Scholar and references therein). The efficiency of cisplatin-DNA lesion removal by NER has been shown to vary among different intrastrand cross-links, and biochemical studies have demonstrated that the major determinant in the efficiency of recognition and repair was the extent of structural distortion of the DNA caused by a given cross-link (14Moggs J.G. Szymkowski D.E. Yamada M. Karran P. Wood R.D. Nucleic Acids Res. 1997; 25: 480-491Crossref PubMed Scopus (117) Google Scholar). It has been suggested that poor substrates for removal by NER, such as 1,2-intrastrand cross-links, tend to undergo replicative bypass, leading to the possible insertion of mispaired bases opposite the cross-links and the intervention of the mismatch repair (MMR) pathway. Consistent with this view, in vitro studies have shown that certain types of mismatched cisplatin-DNA adducts can be specifically recognized by the mismatch recognition complex hMutSα (15Mu D. Tursun M. Duckett D.R. Drummond J.T. Modrich P. Sancar A. Mol. Cell. Biol. 1997; 17: 760-769Crossref PubMed Scopus (179) Google Scholar, 16Yamada M. O'Regan E. Brown R. Karran P. Nucleic Acids Res. 1997; 25: 491-496Crossref PubMed Scopus (141) Google Scholar). These observations may provide an explanation why MMR activity is compromised in certain cell lines selected for their resistance to cisplatin. It should be noted, however, that abrogation of MMR does not seem to occur frequently as a mechanism by which human cells acquire resistance to cisplatin (17Massey A. Offman J. Macpherson P. Karran P. DNA Repair (Amst.). 2003; 2: 73-89Crossref PubMed Scopus (34) Google Scholar). In addition to the specific factors mentioned above, several other proteins, such as members of the high mobility group of proteins involved in chromatin compaction and gene expression, are capable of recognizing distortions of the double helix caused by cisplatin-DNA adducts (1Wozniak K. Blasiak J. Acta Biochim. Pol. 2002; 49: 583-596Crossref PubMed Scopus (132) Google Scholar, 2Zamble D.B. Lippard S.J. Trends Biochem. Sci. 1995; 20: 435-439Abstract Full Text PDF PubMed Scopus (482) Google Scholar). In binding to such lesions, these proteins have the potential to mask the damaged DNA and prevent its repair or to recruit repair machineries, thereby facilitating the removal of the lesions. Hijacking effects, whereby binding of a factor to a cisplatin-DNA lesion is detrimental to other processes normally mediated by this factor, have also been suggested to concur to the cytotoxicity of cisplatin (18Zhai X. Beckmann H. Jantzen H.M. Essigmann J.M. Biochemistry. 1998; 37: 16307-16315Crossref PubMed Scopus (87) Google Scholar). The recognition of cisplatin-DNA lesions therefore appears to contribute greatly to the mechanisms by which cells express resistance to this drug. Here we report a novel gene involved in the cell response to cisplatin in vertebrates. The RDM1 gene (for RAD52 Motif 1) was identified while searching databases for sequences showing similarities to the DNA recombination and repair gene RAD52 (Ref. 19van den Bosch M. Lohman P.H. Pastink A. Biol. Chem. 2002; 383: 873-892Crossref PubMed Scopus (139) Google Scholar and Ref. 20West S.C. Nat. Rev. Mol. Cell. Biol. 2003; 4: 435-445Crossref PubMed Scopus (809) Google Scholar and references therein), by virtue of a small region of aa similarity, hereafter called the RD motif, that the RDM1 protein shares with a functionally important N-terminal region of RAD52. RDM1–/– cells, generated by ablation of RDM1 in the chicken B cell line DT40, exhibited increased sensitivity to cisplatin. However, these cells were not hypersensitive to DNA damages caused by ionizing radiation, UV irradiation, or the alkylating agent methylmethane sulfonate (MMS). The RDM1 protein displays a nucleic-acid binding domain of the RNA recognition motif (RRM) type. By using gel-shift assays and electron microscopy, we show that purified, recombinant chicken RDM1 protein interacts with single-stranded DNA (ssDNA) as well as double-stranded DNA (dsDNA), on which it assembles filament-like structures. Most importantly, RDM1 was found to recognize distortions of the double helix induced by cisplatin-DNA adducts. Finally, high expression of human RDM1 transcripts was detected in the testis, suggesting that RDM1 might play a role during spermatogenesis. DNA and Protein Sequence Analyses—Sequence similarity searches were carried out using the BLAST family of programs (21Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59933) Google Scholar). The RRM motif of RDM1 was identified by using HHMER (22Eddy S.R. Bioinformatics. 1998; 14: 755-763Crossref PubMed Scopus (4067) Google Scholar) to query the PFAM library of Hidden Markov models of protein families (23Bateman A. Birney E. Cerruti L. Durbin R. Etwiller L. Eddy S.R. Griffiths-Jones S. Howe K.L. Marshall M. Sonnhammer E.L. Nucleic Acids Res. 2002; 30: 276-280Crossref PubMed Scopus (2015) Google Scholar). The crystal structure of human RAD52 was examined using Swiss PDB Viewer (24Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9590) Google Scholar). All programs were used with default parameters. Sequence data have been submitted to the DDBJ/GenBank™/EBI Data Bank under accession numbers AB080727 (chicken RDM1) and AB080728 (human RDM1). Cloning of the Chicken RDM1 Gene and Its Targeted Disruption in the Chicken B Cell Line DT40 —The chicken RDM1 cDNA as well as its 5′ and 3′ regions were cloned using the SMART RACE cDNA amplification kit (Clontech). The genomic DNA fragments used to construct the RDM1 knock-out vectors were amplified using long range PCR, from DNA extracted from chicken DT40 cells. Gene disruption vectors (pRdm1Puro and pRdm1Bsr) were constructed as described previously (25Arakawa H. Hauschild J. Buerstedde J.M. Science. 2002; 295: 1301-1306Crossref PubMed Scopus (385) Google Scholar). Recycling of the drug resistance markers was achieved using pLox vectors as described (26Arakawa H. Lodygin D. Buerstedde J.M. BMC Biotechnol. 2001; 1: 7Crossref PubMed Scopus (150) Google Scholar). DT40 cell culture, transfection, and genomic Southern blots were done as described previously (25Arakawa H. Hauschild J. Buerstedde J.M. Science. 2002; 295: 1301-1306Crossref PubMed Scopus (385) Google Scholar). DT40 Colony Survival Assay—The survival of DT40 cells in cisplatin- and MMS-containing medium was measured by plating serially diluted cells in chicken medium (25Arakawa H. Hauschild J. Buerstedde J.M. Science. 2002; 295: 1301-1306Crossref PubMed Scopus (385) Google Scholar) containing 1% methylcellulose. Sensitivity to γ-radiation was measured by exposing plated cells to a 137Cs γ-ray source. UV irradiation (254 nm) was performed on cells suspended in phosphate-buffered saline. Survival is expressed as a percentage, using untreated cells as the 100% value. DNA Substrates and Proteins—Single- and double-stranded circular ϕX174 DNA was purchased from New England Biolabs. Linear ϕX174 DNA with a unique 3′-tail (603 nucleotides in length) was constructed by PstI digestion of gapped circular ϕX174 DNA containing a 603-bp region of ssDNA, which was produced as follows: the 4783-bp PstI-ApaLI restriction fragment of ϕX174 DNA was isolated by centrifugation on a neutral 5–20% sucrose gradient as described previously (27Muller B. West S.C. Methods Mol. Biol. 1994; 30: 413-423PubMed Google Scholar). This fragment was heat-denatured, and the complementary strand was annealed to circular (+) ssDNA of ϕX174, followed by purification of the gapped DNA by agarose gel electrophoresis and electroelution. The gapped DNA was precipitated, resuspended in 10 mm Tris-HCl, pH 8.0, 1mm EDTA (TE), and dialyzed against TE for 24 h at 4 °C. The 52-mer oligonucleotide EV044 used in this study is complementary to nucleotides 130–181 of the single-stranded (+) form of ϕX174 DNA. Gel-purified oligonucleotides were purchased from Proligo. DNA substrates were 5′-32P-end-labeled using polynucleotide kinase and [γ-32P]ATP. Single-stranded ϕX174 DNA was 32P-labeled as described (28Van Dyck E. Hajibagheri N.M. Stasiak A. West S.C. J. Mol. Biol. 1998; 284: 1027-1038Crossref PubMed Scopus (92) Google Scholar). All DNA concentrations are expressed in moles of nucleotides. Cisplatination of XhoI-cut ϕX174 dsDNA was carried out in a 100-μl reaction containing 10 mm Tris-HCl, pH 7.5, 5 mm NaCl, with 5 μg of DNA and appropriate cisplatin:DNA molar ratios by incubating at 37 °C for 16 h in the dark. Unreacted cisplatin was removed by dialysis against TE at 4 °C. Expression and Purification of Recombinant RDM1—Plasmid pEVD008 was constructed by subcloning the chicken RDM1 gene into the Escherichia coli expression vector pET15b (Novagen). Recombinant chicken RDM1 protein was purified from 3.2 liters of E. coli BL21(DE3)trxB– (29Proba K. Ge L. Pluckthun A. Gene (Amst.). 1995; 159: 203-207Crossref PubMed Scopus (67) Google Scholar) (a kind gift from A. Pluckthum and P. Lindner) carrying plasmid pEVD008. Cells were grown at 37 °C in Luria broth containing 60 μg/ml ampicillin and 25 μg/ml kanamycin to an A600 of 0.5 and induced by the addition of 1 mm IPTG, followed by a further 2-h incubation at 30 °C. Cells were harvested by centrifugation and resuspended in 40 ml of T buffer (20 mm Tris-HCl, pH 8.0, 0.5 m NaCl, 10% (v/v) glycerol, 0.02% (v/v) Triton X-100) containing 5 mm imidazole, frozen rapidly in liquid nitrogen, and stored at –80 °C. Bacteria were thawed and lysed by sonication in the presence of phenylmethylsulfonyl fluoride (1 mm), aprotinin (2 μg/ml), and leupeptin (2 μg/ml). Cell debris and insoluble materials were removed by centrifugation at 40,000 rpm for 45 min in a Beckman 50.2 Ti rotor. The supernatant was passed through a 0.45-μm filter and loaded onto a 40-ml Talon (Clontech) column equilibrated with T buffer containing 5 mm imidazole. The column was washed successively with 240 and 120 ml of T buffer containing 5 and 25 mm imidazole, respectively, and RDM1 was eluted with a 400-ml linear gradient of 0.025–1 m imidazole in the same buffer. To prevent possible contaminations from E. coli exonuclease I (30Prasher D.C. Conarro L. Kushner S.R. J. Biol. Chem. 1983; 258: 6340-6343Abstract Full Text PDF PubMed Google Scholar), fractions containing RDM1 (identified by SDS-PAGE) were pooled, dialyzed against R buffer (20 mm Tris-HCl, pH 8.0, 1mm EDTA, 0.5 mm dithiothreitol, 10% glycerol) containing 50 mm KCl, and loaded onto a 14-ml heparin-Sepharose 6 (Amersham Biosciences) column equilibrated with the same buffer. The column was washed with 400 ml of R buffer containing 50 mm KCl before a 400-ml linear gradient of 0.05–1 m KCl in R buffer was applied. Fractions containing RDM1 were dialyzed against R buffer containing 100 mm KCl (R100) and loaded onto a 20-ml Q-Sepharose column (Amersham Biosciences). The column was washed with 100 ml of the same buffer, and the protein was eluted with a 200-ml linear gradient of 0.1–1 m KCl in R buffer. RDM1 was dialyzed against R100 and loaded onto a 1-ml MonoQ (Amersham Biosciences) column equilibrated with the same buffer. The column was washed with R100, and a linear gradient of 0.1–1 m KCl in R buffer was applied. The RDM1 protein, which eluted at ∼350 mm KCl, was dialyzed against R100 and stored at –70 °C. Protein concentrations were determined using the Bio-Rad protein assay kit and bovine serum albumin as standard. The final yield of RDM1 was about 3 mg. Protein dilutions were made in R100 buffer. DNA Binding Assays—Reactions (20 μl) contained the 32P-labeled DNA substrates in a standard binding buffer (20 mm MES, pH 6.4, or 20 mm sodium acetate, pH 6.0, 1 mm dithiothreitol). After 5 min at 37 °C, 1 μl of RDM1 protein (or R100 buffer) was added, and incubation was continued for a further 10 min. Complexes were fixed by addition of glutaraldehyde (final concentration of 0.2% (v/v)) followed by 15 min of incubation at 37 °C. Protein-DNA complexes were resolved by electrophoresis through 0.8% agarose gels run in TAE buffer (or, in the case of oligonucleotide substrates, 10% polyacrylamide gels in TBE buffer), dried onto filter paper, and visualized by autoradiography. In preliminary experiments, no protein-DNA complexes were detected when unfixed reactions with the 52-mer oligonucleotide were analyzed by gel-shift assays, as these were found to dissociate even under conditions of low ionic strength electrophoresis (data not shown). Therefore, glutaraldehyde was added to all the binding reactions analyzed in this study. It should be noted, however, that unfixed complexes assembled on larger substrates (ss and ds ϕX174 DNA) were more stable and resulted in binding patterns identical to those observed with fixed complexes (data not shown). Electron Microscopy—Binding reactions were fixed by addition of glutaraldehyde to 0.2%, followed by 15 min of incubation at 37 °C. Samples were then diluted and washed in 5 mm Mg(OAc)2 before uranyl acetate staining as described previously (31Sogo J. Stasiak A. DeBernardin W. Losa R. Koller T. Sommerville J. Scheer U. Electron Microscopy in Molecular Biology. IRL Press at Oxford University Press, Oxford1987: 61-79Google Scholar). Complexes were observed using a Philips CM100 electron microscope. Northern Blot Analysis—A multiple tissue Northern blot with each lane containing ∼2 μg of poly(A)+ RNA from specific tissues was purchased from Clontech. The membrane was hybridized with a 32P-labeled probe corresponding to the human RDM1 open reading frame according to the manufacturer's instructions. A human β-actin cDNA probe (Clontech) was used as a loading control. Identification and Cloning of the Chicken and Human RDM1 Genes—A database of chicken bursal ESTs has been established as a resource for the analysis of vertebrate gene function (32Abdrakhmanov I. Lodygin D. Geroth P. Arakawa H. Law A. Plachy J. Korn B. Buerstedde J.M. Genome Res. 2000; 10: 2062-2069Crossref PubMed Scopus (69) Google Scholar). While searching this database for candidate genes involved in DNA repair and/or recombination, we identified an EST whose deduced aa sequence displayed partial similarity to an N-terminal region of the RAD52 protein (see below). Rapid amplification of cDNA ends was used to isolate the full-length cDNA of this gene, revealing a single open reading frame (ORF) of 277 aa (Fig. 1A). Further analysis indicated that the sequence similarity between this protein and RAD52 was restricted to a short stretch of aa, hereafter called the RD motif (see below). Because this motif was located in an important region of RAD52, and to reflect its presence in the newly identified gene, we named this gene RDM1 (for RAD52 Motif 1). Exons of the human RDM1 gene were first identified in genomic DNA databases using TBlastN and the chicken gene as query. The complete human RDM1 gene, encoding a 284-aa polypeptide, was subsequently isolated by PCR, both from a testis and a brain cDNA library. Identical ORFs were amplified in both cases (data not shown). The human RDM1 gene is located in region q11.2 of chromosome 17. Finally, a search of SwissProt/TREMBL databases using BLAST identified a mouse protein (Q9CQK3, RIKEN cDNA clone 2410008M22) sharing significant sequence similarity with chicken and human RDM1 (see below). Sequence Analysis of the RDM1 Proteins—A multiple sequence alignment of human (284 aa), mouse (281 aa), and chicken (277 aa) RDM1 proteins is shown in Fig. 1A. Human RDM1 was found to display 71.1 and 51.2% identity and 78.9 and 62.6% similarity, respectively, with its murine and chicken homologs. When used to query the PFAM library of protein domains, the RDM1 sequences revealed the presence, in their N-terminal part, of a nucleic acid-binding motif of the RRM type (Fig. 1B). RRM signatures are found predominantly in various RNA- and ssDNA-binding proteins (34Birney E. Kumar S. Krainer A.R. Nucleic Acids Res. 1993; 21: 5803-5816Crossref PubMed Scopus (589) Google Scholar, 35Ding J. Hayashi M.K. Zhang Y. Manche L. Krainer A.R. Xu R.M. Genes Dev. 1999; 13: 1102-1115Crossref PubMed Scopus (283) Google Scholar, 36Varani G. Nagai K. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 407-445Crossref PubMed Scopus (252) Google Scholar), but their interaction with duplex DNA has also been reported (37DeAngelo D.J. DeFalco J. Rybacki L. Childs G. Mol. Cell. Biol. 1995; 15: 1254-1264Crossref PubMed Google Scholar). It is interesting to note that in human RDM1, the RRM motif corresponds exactly to exon 2 (data not shown). We next investigated the sequence similarity between the RDM1 and RAD52 proteins. Human and yeast RAD52 proteins form multimeric ring structures that interact with ssDNA to mediate single-strand annealing (SSA) as well as RAD51-dependent and -independent strand invasion mechanisms (Refs. 20West S.C. Nat. Rev. Mol. Cell. Biol. 2003; 4: 435-445Crossref PubMed Scopus (809) Google Scholar and 38Kagawa W. Kurumizaka H. Ikawa S. Yokoyama S. Shibata T. J. Biol. Chem. 2001; 276: 35201-35208Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar and references therein, 39Stasiak A.Z. Larquet E. Stasiak A. Muller S. Engel A. Van Dyck E. West S.C. Egelman E.H. Curr. Biol. 2000; 10: 337-340Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar and 40Sung P. Krejci L. Van Komen S. Sehorn M.G. J. Biol. Chem. 2003; 278: 42729-42732Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). It has been proposed that the ssDNA bound by RAD52 is exposed around the outside of the ring (41Parsons C.A. Baumann P. Van Dyck E. West S.C. EMBO J. 2000; 19: 4175-4181Crossref PubMed Scopus (72) Google Scholar, 42Van Dyck E. Stasiak A.Z. Stasiak A. West S.C. EMBO Rep. 2001; 2: 905-909Crossref PubMed Scopus (60) Google Scholar). Recently, the crystal structure of RAD52 variants proficient in SSA was solved (43Kagawa W. Kurumizaka H. Ishitani R. Fukai S. Nureki O. Shibata T. Yokoyama S. Mol. Cell. 2002; 10: 359-371Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 44Singleton M.R. Wentzell L.M. Liu Y. West S.C. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13492-13497Crossref PubMed Scopus (175) Google Scholar), providing further insights into the mechanisms by which RAD52 rings interact with DNA. In a preliminary analysis, the sequence similarity shared by the RDM1 and RAD52 proteins was found to be restricted to an N-terminal region of RAD52 containing its ssDNA binding and ring formation domains. We next analyzed representative sequences of both families using MEME, a program that detects over-represented sequence motifs in unaligned sequences (45Bailey T.L. Elkan C. Proc. Int. Conf. Intell. Syst. Mol. Biol. 1994; 2: 28-36PubMed Google Scholar, 46Bailey T.L. Gribskov M. Bioinformatics. 1998; 14: 48-54Crossref PubMed Scopus (930) Google Scholar). A statistically significant (E value 3.5 × 10–75) 29-residue-long sequence motif shared by all sequences was identified (Fig. 1C). This motif, which we have named RD motif, spans residues 101–130 in chicken RDM1 and residues 104–133 in human and mouse RDM1. The precise location of ssDNA at the surface of a RAD52 ring is still unknown for lack of a co-crystal. However, the ssDNA is most probably bound within a deep groove that runs continuously around the outside of the ring (44Singleton M.R. Wentzell L.M. Liu Y. West S.C. Wigley D.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13492-13497Crossref PubMed Scopus (175) Google Scholar). We noted that the N-terminal strand of the identified motif (aa 61–65) was part of a hairpin structure that comprises part of the proposed DNA-binding groove (Fig. 1D). Other residues of the RD motif take part in the interaction between the RAD52 monomers. For example, Phe-79 whose aromatic character is conserved in both RAD52 and RDM1 proteins forms a hydrophobic cluster with aa Leu-115, Tyr-81, Phe-158, and Tyr-31 of the adjacent monomer. Likewise, residues of helix 69–78 are in contact with residues of the adjacent monomer. Finally, we noted the presence of Cys-127 in the RD motif of human RDM1, although a Trp was present at this position in the other proteins of Fig. 1C. However, databases of single nucleotide polymorphisms report a Cys/Trp polymorphism at this position in human RDM1, with an allele frequency of ∼0.8 T (Cys) and ∼ 0.2 G (Trp) (NCBI SNP cluster ID, rs2251660; JSNP ID, IMS-JST047036). Disruption of RDM1 in Chicken DT40 Cells Confers Increased Sensitivity to Cisplatin—To investigate the role of RDM1 in DNA repair and recombination, we decided to disrupt it in the chicken B cell line DT40, whose genome can easily be modified by targeted recombination. To this end, we used a DT40 clone (DT40Cre1) containing a tamoxifen-inducible Cre recombinase (MerCreMer) gene that can excise loxP-flanked (floxed) cassettes. Two RDM1 knock-out constructs (pRdm1Bsr and pRdm1Puro, harboring a blasticidin and a puromycin resistance marker, respectively) were made by cloning genomic fragments of the chicken RDM1 locus upstream and downstream of floxed drug resistance markers (Fig. 2A). Targeted integration of these constructs was expected to delete nearly half of the RDM1 coding region (from codon 66 to 181). Following transfection of pRdm1Bsr into DT40Cre1, a heterozygous RDM1 clone was identified (DT40 RDM1+/–), which was subsequently transfected by pRdm1Puro to produce a homozygous RDM1 knock-out clone (DT40 RDM1–/–) (Fig. 2A), indicating that RDM1 is not an essential gene. Both the blasticidin and the puromycin resistance markers were then removed from DT40 RDM1–/– by induction of the Cre recombinase, yielding the clone DT40 RDM1–/–E (where E indicates excised). The disruption of RDM1 and the excision of the drug resistance marker were confirmed by Southern blot analysis (Fig. 2B). The overall growth of RDM1–/–E cells was found to be comparable with that of wild-type cells. To examine the importance of RDM1 for DNA repair, we examined the viability of cells challenged with a variety of DNA-damaging agents using colony survival assays. For comparison purposes, we also used a DT40 cell line deficient in RAD54, a gene involved in HR and DSB repair (11Dronkert M.L. Kanaar R. Mutat. Res. 2001; 486: 217-247Crossref PubMed Scopus (487) Google Scholar, 47Bezzubova O. Silbergleit A. Yamaguchi-Iwai Y. Takeda S." @default.
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• W2022066102 title "RDM1, a Novel RNA Recognition Motif (RRM)-containing Protein Involved in the Cell Response to Cisplatin in Vertebrates" @default.
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• W2022066102 doi "https://doi.org/10.1074/jbc.m412874200" @default.
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