- Email: [email protected]
Sequence of mouse CDC47, a member of the minichromosome maintenance (Mem) family involved in the DNA replication licensing system
Gene. 167 11995)343-344 © 1095 ElsevierScienceB.V.All riots reserved.0378-1119/95/$09.50
343
GENE 09417
Sequence of mouse CDC47, a member of the minichromosome maintenance (Mcm) family involved in the DNA replication licensing system (Cell proliferation; DNA-dependent ATPase; zinc finger)
N a n a k o T a k i z a w a a, H i r o s h i K i m u r a b a n d K a z u n o r i S u g i m o t o a aDepartment of Chemistr¢ 11. Facultyof Scwnce. and bResearchCenterfor Molecular Genetics. Hokkaido Unit:ersit3;Sapporo060, Japan
Received by H. Yoshikawa:21 July 1995;Revised/Accepted:23 August/26August1995;Receivedat publishers:2 October 1995
SUMMARY The cDNA encoding a mouse protein, mCDC47, belonging to the minichromosome maintenance (Mcm) family possibly involved in the DNA replication licensing system, was cloned and sequenced. Its deduced amino acid (aa) sequence contained a putative DNA-dependent ATPase motif commonly observed in the family, and a zinc-finger-like domain found in some members of the family.
Minichromosome maintenance (Mcm) proteins are considered to be involved in the DNA replication licensing system as components of licensed chromatin. They bind to chromatin at the M phase and are released from replicated chromatin during the S phase in yeast (Tye, 1994) and mammalian cells (Kimura et al., 1994). Their essential role in the initiation of DNA replication was shown by yeast gene disruption analyses (T2,e, 1994), an in vitro replication system of Xenopus egg extract~ (Kubota et al., 1995) and microinjection of the spec;_fic antibodies into the nucleus of mammalian cells (Kimura et al., 1994; Todorov et al., 1994). The genetic and the protein-protein interactions observed among the Mcm family consisted of at least six members (Tye, 1994;
Correspondence to: Dr. tt. Kimura, Research Center for Molecular Genetics, HokkaidoUniversity,Kita 10Nishi 8, Kita-ku.Sapporo060. Japan. Tel. (81-11) 706-2798; Fax (81-11) 737-0536; e-mail: [email protected]
Abbreviations: aa, amino acid(s);bp, base pair(s); CDC, cell division cycleprotein;CDC, goneencodingCDC;kb, kilobase(s)or 1~,300bp; m-, mouse; Mcm, minichromosomemaintenance {protein); Mcm, gone encoding Mere; ut, nucleotidels); PCR, polymerasechain reaction; UTR, untranslated regionls);X, any aa; Zf, zinc finger(s). SSDI 0378-1119(95)00713-X
Kimura et al., 1995) suggest that Mcm proteins cooperatively function for the initiation of DNA replication. We have isolated three mouse Mcm-related eDNA clones, P I M C M 3 , mCDC46 and mCdc21, putative homologs of Saccharomyces cerevisiae Mcm3, CDC46/Mcm5 and Schizosaccharmnyces pomhe Cdc21 (Kimura et al., 19951. The partial nt sequence of a new Mere-related gone was amplified using a forward (5'-CCGAATTCGAYCCWGGHGCYAARTC) and a reverse ( 5'-CCAAGCTTATCTTRTCRAAYTCRTCRAAYTCRTCDAT) primer (where D = A or G or T; H = A or C or 1"; R = A or G; W = A or T; Y = C or T) corresponding to the aa sequences DPGTAKS and IDEFDKM, from a ~,gt10 FM3A eDNA library by PCR. The eDNA was then isolated by screening of the library with the PCR product and the nt sequence was determined (Fig. 1). The primer extension analysis showed that this 2.4-kb eDNA was nearly full length. The deduced polypeptide consisted of 719 aa (81.5 kDa). In the central region tile putative DNA-dependent ATPase motif which is highly conserved throughout Mere-related proteins (Hu et al., 1993) was found. The consensus aa sequence found in all Mcm members (IDEFDKM) was also conserved in this new mouse Mcm protein (Fig. 1).
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Fig. L Thc nt seqocnee of mCDC47(DDBJ accession No. D26091) and the deduced aa sequence. The stop codon (TGA) is indicated by an asterisk and the polyadenylation signal (AATAAA)in the 3' UTR is shown by a thick u,~der[i~,c.The aa region of putative ATPase motif is underlined and the consensus aa sequencxamong all Mcm proteins (IDEFDKM) is shown in boldface. The Cys rcsidt:es possibly associated with the metal binding arc shown in italicizcd boldface. This mouse protein has 4 4 % a a identity with budding yeast YB52, the possible product of an open reading frame, yBRI441, found by systematic sequencing of chromosome II (Bussereau et al., 1993), but has only 2 6 - 3 0 % identity with other yeast M c m proteins. Similar considerable similarity has been found between yeast and mouse homologs of other Mcm proteins, Mcm3 (42%), C D C 4 6 (47%) and Cdc21 (44%). Since yBRI441 was recently shown to be identical to CDC47 (Dalton and Whitbread, 1995), ':he mouse gene we cloned was named as mCDC47. The possible h u m a n homolog of mCDC47, reported as Pl.lMcm3 (Hu et al., 1993) or MCM2 ( N a k a t s u r u et al., 1994), shows a b o u t 95% identity at the a a level with mCDC47. Since h u m a n Mcm2 h o m o l o g has already been reported as BM28 (Todorov et al., 1994) and CDC47 is different from Mcm3 (Dalton and Whitbread, 1995), the h u m a n gene (PI.I Mcm3 and "MCM2') should be renamed as PICDC47 according to the nomenclature of h u m a n Mcm family (Hu et al., 1993) to avoid confusion. t-aCDC47 has a Zf-like structure which is commonly observed in Mcm proteins except Mcm3 and C D C 4 6 (Fig. 1). This putative Zf (CX,.CX~RCX4C) is similar to that found in Mis5 and Cdc21 but different from Zf found in Mcm2 (CXzCXtgCX,C). The a a sequence ht Zf region is conserved in Mcm proteins relatively well, but not in C D C 4 6 and Mcm3, suggesting its important role. In fact, one of the Cys residues is essential for the function of yeast Mcm2 and it is suggested that this region is involved in the protein-protein interaction (Tye, 1994).
ACKNOWLEDGEMENTS We t h a n k Dr. N o b u o Takagi for reading the m a n u script, Dr. Takashi Sado for the mouse e D N A library and
Dr. Sanae M i y a k e for the PCR primers. This study was partly supported by Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.
REFERENCES Bussereau. F.. Mallet, L., Gaillon. L. and Jacquet, M.: A 12.8kb segment, on the fight arm of chromosome 1I from Saccharomyces cereeisiae including part of the DURI. 2 gene, contains five putative new genes. Yeast 9 {1993) 797-806. Dalton, S. and Whitbrcad. L.: Cell cycle-regulatedimport of Cdc47, a protein essential for initiation of DNA replication in budding yeast. Proc. Natl. Acad. Sci. USA 92 (1995l 2514-2518. Hu. B., Burkhart, R., Schulte, D., Musahl, C. and Knippers, R.: The P1 family: anew class of nuclear mammalian proteins related to the yeast Mcm replication proteins. Nucleic Acids Res. 21 (1993) 5289-5293. Kimura. H. Nozaki, N. and Sugimoto, K.: DNA polymerase a associated protein Pl, a mufin¢ homolog of yeast MCM3. changes its intranuclear distribution during the DNA synthetic period. EMBO J. 13 (1994) 4311-4320. Kimura, H., Takizawa, N., Nozaki, N. and Sugimoto. K.: Molecular cloning of eDNA encoding mouse Cdc21 and CDC46 homologs and characterization of the products: physical interaction between PI(MCM3) and CDC46 proteins. Nucleic Acids Res. 23 {1995) 2097-2104. Kubota, Y., Miura, S., Nishimoto. S.-i., Takisawa, H. and Noj~ma, H.: Identification of the yeast MCM3-related protein as a component of Xeaopas DNA replication licensing factor. Cell 81 (1995) 601-609. Nakatsuru, S., Sudo, K. and Nakamura, Y.: Isolation and mapping of human gene (MCM2) encoding a product homologous to ye.~st proteins invoh'ed in DNA replication. Cytogenet. Cell C,=oct. 68 (1995) 226-230. Todorov, I.T., Pepperkok, R., Philipova, R.N.. Kea=sey,S., Ansorge,W. and Werner, D.: A human nuclear protein with sequence homology to a family of early S phase proteins is required for the entry into S phase and for cell division. J. Cell Sci. 107 (1994) 253-265. Tye, B.-K.: The Mcm2-3-5 proteins: are they replication licensing factors? "FiendsCell Biol. 4 (1994) 160-166.
343
GENE 09417
Sequence of mouse CDC47, a member of the minichromosome maintenance (Mcm) family involved in the DNA replication licensing system (Cell proliferation; DNA-dependent ATPase; zinc finger)
N a n a k o T a k i z a w a a, H i r o s h i K i m u r a b a n d K a z u n o r i S u g i m o t o a aDepartment of Chemistr¢ 11. Facultyof Scwnce. and bResearchCenterfor Molecular Genetics. Hokkaido Unit:ersit3;Sapporo060, Japan
Received by H. Yoshikawa:21 July 1995;Revised/Accepted:23 August/26August1995;Receivedat publishers:2 October 1995
SUMMARY The cDNA encoding a mouse protein, mCDC47, belonging to the minichromosome maintenance (Mcm) family possibly involved in the DNA replication licensing system, was cloned and sequenced. Its deduced amino acid (aa) sequence contained a putative DNA-dependent ATPase motif commonly observed in the family, and a zinc-finger-like domain found in some members of the family.
Minichromosome maintenance (Mcm) proteins are considered to be involved in the DNA replication licensing system as components of licensed chromatin. They bind to chromatin at the M phase and are released from replicated chromatin during the S phase in yeast (Tye, 1994) and mammalian cells (Kimura et al., 1994). Their essential role in the initiation of DNA replication was shown by yeast gene disruption analyses (T2,e, 1994), an in vitro replication system of Xenopus egg extract~ (Kubota et al., 1995) and microinjection of the spec;_fic antibodies into the nucleus of mammalian cells (Kimura et al., 1994; Todorov et al., 1994). The genetic and the protein-protein interactions observed among the Mcm family consisted of at least six members (Tye, 1994;
Correspondence to: Dr. tt. Kimura, Research Center for Molecular Genetics, HokkaidoUniversity,Kita 10Nishi 8, Kita-ku.Sapporo060. Japan. Tel. (81-11) 706-2798; Fax (81-11) 737-0536; e-mail: [email protected]
Abbreviations: aa, amino acid(s);bp, base pair(s); CDC, cell division cycleprotein;CDC, goneencodingCDC;kb, kilobase(s)or 1~,300bp; m-, mouse; Mcm, minichromosomemaintenance {protein); Mcm, gone encoding Mere; ut, nucleotidels); PCR, polymerasechain reaction; UTR, untranslated regionls);X, any aa; Zf, zinc finger(s). SSDI 0378-1119(95)00713-X
Kimura et al., 1995) suggest that Mcm proteins cooperatively function for the initiation of DNA replication. We have isolated three mouse Mcm-related eDNA clones, P I M C M 3 , mCDC46 and mCdc21, putative homologs of Saccharomyces cerevisiae Mcm3, CDC46/Mcm5 and Schizosaccharmnyces pomhe Cdc21 (Kimura et al., 19951. The partial nt sequence of a new Mere-related gone was amplified using a forward (5'-CCGAATTCGAYCCWGGHGCYAARTC) and a reverse ( 5'-CCAAGCTTATCTTRTCRAAYTCRTCRAAYTCRTCDAT) primer (where D = A or G or T; H = A or C or 1"; R = A or G; W = A or T; Y = C or T) corresponding to the aa sequences DPGTAKS and IDEFDKM, from a ~,gt10 FM3A eDNA library by PCR. The eDNA was then isolated by screening of the library with the PCR product and the nt sequence was determined (Fig. 1). The primer extension analysis showed that this 2.4-kb eDNA was nearly full length. The deduced polypeptide consisted of 719 aa (81.5 kDa). In the central region tile putative DNA-dependent ATPase motif which is highly conserved throughout Mere-related proteins (Hu et al., 1993) was found. The consensus aa sequence found in all Mcm members (IDEFDKM) was also conserved in this new mouse Mcm protein (Fig. 1).
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Fig. L Thc nt seqocnee of mCDC47(DDBJ accession No. D26091) and the deduced aa sequence. The stop codon (TGA) is indicated by an asterisk and the polyadenylation signal (AATAAA)in the 3' UTR is shown by a thick u,~der[i~,c.The aa region of putative ATPase motif is underlined and the consensus aa sequencxamong all Mcm proteins (IDEFDKM) is shown in boldface. The Cys rcsidt:es possibly associated with the metal binding arc shown in italicizcd boldface. This mouse protein has 4 4 % a a identity with budding yeast YB52, the possible product of an open reading frame, yBRI441, found by systematic sequencing of chromosome II (Bussereau et al., 1993), but has only 2 6 - 3 0 % identity with other yeast M c m proteins. Similar considerable similarity has been found between yeast and mouse homologs of other Mcm proteins, Mcm3 (42%), C D C 4 6 (47%) and Cdc21 (44%). Since yBRI441 was recently shown to be identical to CDC47 (Dalton and Whitbread, 1995), ':he mouse gene we cloned was named as mCDC47. The possible h u m a n homolog of mCDC47, reported as Pl.lMcm3 (Hu et al., 1993) or MCM2 ( N a k a t s u r u et al., 1994), shows a b o u t 95% identity at the a a level with mCDC47. Since h u m a n Mcm2 h o m o l o g has already been reported as BM28 (Todorov et al., 1994) and CDC47 is different from Mcm3 (Dalton and Whitbread, 1995), the h u m a n gene (PI.I Mcm3 and "MCM2') should be renamed as PICDC47 according to the nomenclature of h u m a n Mcm family (Hu et al., 1993) to avoid confusion. t-aCDC47 has a Zf-like structure which is commonly observed in Mcm proteins except Mcm3 and C D C 4 6 (Fig. 1). This putative Zf (CX,.CX~RCX4C) is similar to that found in Mis5 and Cdc21 but different from Zf found in Mcm2 (CXzCXtgCX,C). The a a sequence ht Zf region is conserved in Mcm proteins relatively well, but not in C D C 4 6 and Mcm3, suggesting its important role. In fact, one of the Cys residues is essential for the function of yeast Mcm2 and it is suggested that this region is involved in the protein-protein interaction (Tye, 1994).
ACKNOWLEDGEMENTS We t h a n k Dr. N o b u o Takagi for reading the m a n u script, Dr. Takashi Sado for the mouse e D N A library and
Dr. Sanae M i y a k e for the PCR primers. This study was partly supported by Grants-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan.
REFERENCES Bussereau. F.. Mallet, L., Gaillon. L. and Jacquet, M.: A 12.8kb segment, on the fight arm of chromosome 1I from Saccharomyces cereeisiae including part of the DURI. 2 gene, contains five putative new genes. Yeast 9 {1993) 797-806. Dalton, S. and Whitbrcad. L.: Cell cycle-regulatedimport of Cdc47, a protein essential for initiation of DNA replication in budding yeast. Proc. Natl. Acad. Sci. USA 92 (1995l 2514-2518. Hu. B., Burkhart, R., Schulte, D., Musahl, C. and Knippers, R.: The P1 family: anew class of nuclear mammalian proteins related to the yeast Mcm replication proteins. Nucleic Acids Res. 21 (1993) 5289-5293. Kimura. H. Nozaki, N. and Sugimoto, K.: DNA polymerase a associated protein Pl, a mufin¢ homolog of yeast MCM3. changes its intranuclear distribution during the DNA synthetic period. EMBO J. 13 (1994) 4311-4320. Kimura, H., Takizawa, N., Nozaki, N. and Sugimoto. K.: Molecular cloning of eDNA encoding mouse Cdc21 and CDC46 homologs and characterization of the products: physical interaction between PI(MCM3) and CDC46 proteins. Nucleic Acids Res. 23 {1995) 2097-2104. Kubota, Y., Miura, S., Nishimoto. S.-i., Takisawa, H. and Noj~ma, H.: Identification of the yeast MCM3-related protein as a component of Xeaopas DNA replication licensing factor. Cell 81 (1995) 601-609. Nakatsuru, S., Sudo, K. and Nakamura, Y.: Isolation and mapping of human gene (MCM2) encoding a product homologous to ye.~st proteins invoh'ed in DNA replication. Cytogenet. Cell C,=oct. 68 (1995) 226-230. Todorov, I.T., Pepperkok, R., Philipova, R.N.. Kea=sey,S., Ansorge,W. and Werner, D.: A human nuclear protein with sequence homology to a family of early S phase proteins is required for the entry into S phase and for cell division. J. Cell Sci. 107 (1994) 253-265. Tye, B.-K.: The Mcm2-3-5 proteins: are they replication licensing factors? "FiendsCell Biol. 4 (1994) 160-166.