The Essential Role of Saccharomyces cerevisiae CDC6 Nucleotide-binding Site in Cell Growth, DNA Synthesis, and Orc1 Association

Abstract

Saccharomyces cerevisiaeCdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition. Saccharomyces cerevisiaeCdc6 is a protein required for the initiation of DNA replication. The biochemical function of the protein is unknown, but the primary sequence contains motifs characteristic of nucleotide-binding sites. To study the requirement of the nucleotide-binding site for the essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We have used these mutants to investigate in vivo roles of the conserved lysine in the growth rate of transformant cells and the complementation of cdc6temperature-sensitive mutant cells. Our results suggest that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss-of-function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to wild-type, a silent mutation. To investigate what leads to the growth defects derived from the mutations at the nucleotide-binding site, we evaluated its gene functions in DNA replication by the assays of the plasmid stability and chromosomal DNA synthesis. Indeed, the K114P and K114E mutants showed the complete retraction of DNA synthesis. In order to test its effect on the G1/S transition of the cell cycle, we have carried out the temporal and spatial studies of yeast replication complex. To do this, yeast chromatin fractions from synchronized culture were prepared to detect the Mcm5 loading onto the chromatin in the presence of the wild-type Cdc6 or mutant cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the association with chromatin and in the loading of Mcm5 onto chromatin origins. To further investigate the molecular mechanism of nucleotide-binding function, we have demonstrated that the Cdc6 protein associates with Orc1 in vitro and in vivo. Intriguingly, the interaction between Orc1 and Cdc6 is disrupted when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional ATP-binding of Cdc6, which may be a prerequisite step to assemble the operational replicative complex at the G1/S transition. cyclin-dependent kinase autonomously replicating sequence cell division cycle glutathioneS-transferase pulsed field gel electrophoresis phenylmethylsulfonyl fluoride replication complex kilobase pair(s) hemagglutinin low phosphate medium fluorescence-activated cell sorting Cell cycle regulation is a complicated but highly coordinated process. It has a conserved mechanism among eukaryotes from yeast to human. The primary control of the eukaryotic cell cycle is provided by a family of cyclin-dependent kinases (CDKs)1 and their associated cyclins, which regulate kinase activity. In unicellular yeast cells, a single prototype CDK gene, CDC28 in the budding yeastSaccharomyces cerevisiae or cdc2 + in the fission yeast Schizosaccharomyces pombe functions at different cell cycle stages. Different cyclins activate the same kinase as different points in the cycle. It is now known that the central control of cell cycle progression by CDK complexes is regulated positively and negatively to monitor each step of the progression (1Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1769) Google Scholar,2Arellano M. Moreno S. Int. J. Biochem. Cell Biol. 1997; 29: 559-573Crossref PubMed Scopus (166) Google Scholar). This regulatory control, associated with checkpoints, orchestrates various types of cell cycle genes throughout the cell cycle. In S. cerevisiae, over 70 temperature-sensitive cell division cycle (cdc) mutations have been isolated that control events throughout the cell cycle (3Pringle J.R. Hartwell L.H. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1981: 97-142Google Scholar, 4Wheals A.E. The Yeasts. Academic Press, London1987: 284-390Google Scholar). One of them,CDC6, is required in the late G1 and S phases of the cell cycle. At the non-permissive temperature, the cdc6mutant cells show a DNA synthesis defect (5Culotti J. Hartwell L.H. Exp. Cell Res. 1971; 67: 389-401Crossref PubMed Scopus (130) Google Scholar). The cdc6mutants undergo increased chromosomal loss and hyper-recombination, suggesting a fairly direct role in DNA replication (6Hartwell L.H. Smith D. Genetics. 1985; 110: 381-395Crossref PubMed Google Scholar). Tandem copies of different ARSs added to the mini-chromosome can suppress mitotic loss in cdc6, but not in cdc7, cdc9,cdc16, or cdc17 mutants (7Hogan E. Koshland D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3098-3102Crossref PubMed Scopus (132) Google Scholar), suggesting a role at replication origins. The CDC6 mRNA fluctuates periodically throughout the cell cycle (8Zhou C. Jong A. J. Biol. Chem. 1990; 265: 19904-19909Abstract Full Text PDF PubMed Google Scholar, 9Piatti S. Lengauer C. Nasmyth K. EMBO J. 1995; 14: 3788-3799Crossref PubMed Scopus (333) Google Scholar, 10Zwerschke W. Rottjakob H.-W. Kuntzel H. J. Biol. Chem. 1994; 269: 23351-23356Abstract Full Text PDF PubMed Google Scholar), and its nuclear entry is cell cycle-dependent (11Jong A.Y. Young M. Chen G.C. Zhang S.Q Chen C. DNA Cell Biol. 1996; 15: 883-895Crossref PubMed Scopus (19) Google Scholar). The of the CDC6 gene is to a of cell cycle genes that for DNA synthesis or in DNA replication C. Jong A.Y. DNA Cell Biol. PubMed Scopus Google Scholar). The of Cdc6 in DNA replication has been by direct of function in mutants gel C. M. 1995; Full Text PDF PubMed Scopus Google Scholar). The studies suggest that the Cdc6 and protein and the of initiation of DNA replication in interaction between CDC6 and has been PubMed Scopus Google Scholar). The S. pombe is to S. cerevisiae CDC6 A. Full Text PDF PubMed Scopus Google Scholar, H. 1995; Full Text PDF PubMed Scopus Google Scholar). It has been that the gene roles in the cell cycle, the initiation of DNA replication and In S. cerevisiae, of the cell cycle A. EMBO J. 1992; PubMed Scopus Google Scholar). results suggest a function of CDC6 at the of yeast Cdc6 have been found in Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google and 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The of Cdc6 among essential role for has cell cycle the of replication complexes in G1 and S to A. 1994; Full Text PDF PubMed Scopus Google Scholar, A. J. EMBO J. 1995; 14: PubMed Scopus Google Scholar, 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The that in and which and The is by its further the of in to the and The suggest that the initiation of DNA replication is not by the of and to the of has to with the of The Cdc6 protein is required for and of the mutant a J. Cell Sci. 1997; 110: Google Scholar, S. C. Nasmyth K. 1996; PubMed Scopus Google Scholar, C. EMBO J. 1996; 15: PubMed Scopus Google Scholar). The Cdc6 protein is required for the loading of D. Nasmyth K. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Dev. 1997; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Google Scholar). In the Cdc6 protein with protein complex is at late G1 S. Jong A. Biol. 1996; PubMed Scopus Google Scholar). We have found that the Cdc6 can to the of DNA Jong A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is that of Cdc6 with a of other protein as the the and kinase in replication and cell cycle control A. Annu. Rev. Dev. Biol. 1997; 13: PubMed Scopus Google Scholar). the molecular mechanism of the is is a conserved nucleotide-binding site in Cdc6 C. Jong A.Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). It has been that the yeast Cdc6 is to and W. Rottjakob H.-W. Kuntzel H. J. Biol. Chem. 1994; 269: 23351-23356Abstract Full Text PDF PubMed Google Scholar). At the roles of the nucleotide-binding site One to study the function of the nucleotide-binding site is to out to the Lys and its In these we have a of to the functions of the nucleotide-binding we changed the conserved Lys114 at the nucleotide-binding site to five other amino acid residues. We show that replacement of Lys with Glu (K114E) and Pro (K114P) leads to loss of function in supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu (K114L) yields partially functional proteins, and replacement with Arg yields a phenotype equivalent to on the plasmid stability and chromosomal DNA synthesis the roles of the conserved Lys is required for DNA replication. This to investigate the of a functional replication complex at G1/S transition. we have prepared chromatin fractions of mutant and wild-type to the effect of the cdc6(K114E) mutation. We found that the loading of the cdc6(K114E) and Mcm5 is in the at the molecular we demonstrated that Cdc6 with Orc1 in vitro and in vivo. the interaction between Orc1 and Cdc6 is when the cdc6(K114E) protein is used. Our results suggest that a proper molecular interaction between Orc1 and Cdc6 depends on the functional of This may to assemble the operational replicative complex at the G1/S transition. The of the in is in The has been S. Jong A. Biol. 1996; PubMed Scopus Google Scholar). with out S. Lengauer C. Nasmyth K. EMBO J. 1995; 14: 3788-3799Crossref PubMed Scopus (333) Google Scholar). is a at lysine were by a as and the A. PubMed Scopus Google Scholar). has been changed to Glu Gln Arg Pro and Leu (K114L) The and for the of were prepared by the wild-type and K114E the control of the in a yeast with as is the same that is the is derived from a and contains a sequence of the CDC6 gene C. Jong A.Y. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the CDC6 in which the of CDC6 gene been and the is by a a in which Mcm5 is at its amino and the of the gene is by its sequence A. Chen E. D. Dev. PubMed Scopus Google Scholar). of for the of protein contains and at the of the gene by the with is the with the of on the with as the The wild-type and temperature-sensitive genes were for Yeast and medium were from were from and were from Life or of the wild-type and CDC6 gene cells at the and as of in a of mutant and wild-type Cdc6 in the the protein from Life and CDC6 the control of as We or copies of the wild-type or mutant by or and were in and added for cells were of each and and the with Cdc6 and CDC6 and and between Orc1 and protein on the used as a protein The same the Orc1 used as control Yeast were prepared from and used as the Cdc6 protein The were the and to protein The of Cdc6 by E. and were used for interaction as of and by that were the same yeast with The with or or with of culture with for The Orc1 by and with on the protein The can detect not protein but is interaction between Orc1 and Cdc6 proteins. The the of Orc1 and the of as of in used to out the as The and were C. Jong A.Y. DNA Cell Biol. PubMed Scopus Google Scholar, Smith M. PubMed Scopus Google DNA of the of DNA used in the C. Jong A. 1990; Google Scholar). The DNA is in Yeast chromosomal The wild-type and mutant were to their plasmid on medium in of used to of or and were at with for of the were on and were to the of cells. were to to the of cells. The used to investigate yeast chromosomal DNA synthesis A.Y. Zhang Biochem. 1995; PubMed Scopus Google Scholar). In yeast cells with in vivo and chromosomal DNA is by pulsed field gel of yeast cell culture at to the and by for The cells were with of low phosphate medium and the same of of The culture for and with The cells were and with and were the DNA field electrophoresis The gel and Yeast chromatin prepared to and Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google with yeast cells plasmid with of and or The transformant cells were on the medium in the presence of to the The cells were synchronized at G1 with at of at for The cells were from on the and at the of cells were at different to monitor the of by of of cells and C. Jong A. 1990; Google Scholar). Yeast cells were with and in a at for the cells were and with were by the cells with yeast in a at for The were on for at for at and with the The were by and in a fluoride and with a Cell were by at for at were and were at and for were in of the and to at and for were in a and and at of protein from each chromatin were with loading and in a for The were by gel electrophoresis at for and were onto The protein were with for proteins, or with for proteins, by The protein were with the of culture protein and of in the presence of and of and of were added and at for The at for The and with of at for The onto a and with of The for Cdc6 interaction were prepared from of were in with and used for gel electrophoresis to their The for the interaction Orc1 and Cdc6 yeast with The with or or with of culture with for The cell were disrupted by in with of Orc1 by and with on protein can detect and on the same Orc1 can in the in C. In vitro is a to the between the and of proteins, the amino acid for a function can be D. D. 1985; PubMed Scopus Google Scholar). of the primary sequence C. Jong A.Y. J. Biol. Chem. Full Text PDF PubMed Google has that a conserved among known from This a role in the of and the Lys is essential for interaction between the and the W. 1985; PubMed Scopus Google Scholar). In order to the conserved Lys essential amino acid as in other nucleotide-binding proteins, we have the Lys to five other residues. The were to the wild-type sequence for or at the in the lysine and The were at the conserved to to test the effect of a to to test the effect of a to to test the effect of to to test the effect of a amino and to to test the effect of the of the Lys and Arg amino of the nucleotide-binding known of for lysine results in a protein with function in in yeast found to its and DNA but not to the to D. S. EMBO J. PubMed Scopus Google Scholar). by DNA To test the in vivo function of the nucleotide-binding site, we the mutant genes can the growth We mutant with the mutant were at the in the wild-type and and to the defective mutant cells at and to The for K114E and K114P to the between the and chromosomal results that the Lys to Glu may the interaction between Cdc6 and the Lys to Pro may in the loss gene the that the Lys is essential for CDC6 gene of the is that the mutant not To that of the mutant to to a defective Cdc6 and not to of we the of wild-type Cdc6 and the cdc6(K114E) proteins. The cdc6(K114E) protein the defect in the complementation Cdc6 is at low in yeast and is with the Cdc6 we the wild-type and mutant the control of and yeast with or and of of cells the were with Cdc6 in mutant and wild-type were at the same that were not the of the of in vivo function of cdc6(K114E) To further the effect of mutant in the with the mutant the wild-type plasmid and and were at cells were to at the to the and for which growth were the wild-type plasmid at a rate to wild-type in medium mutants and cells to and at The wild-type, with of for and for and at of the mutants show at in growth that lysine is for results can be from the at and were in the K114E and K114P the growth of the K114E and K114P to cells with is that the K114E and K114P loss-of-function that the plasmid loss at the temperature, but may be by the of a functional on the We the for their to stability on in cells a plasmid with the wild-type CDC6 gene of plasmid a loss rate to wild-type The plasmid stability and and in cells K114E and K114P show plasmid to that of The loss of plasmid stability is to the of DNA synthesis. To what mechanism the growth rate and plasmid DNA we the chromosomal DNA synthesis. The is a which is highly to chromosomal DNA synthesis in In yeast cells by in vivo and chromosomal DNA by pulsed field gel DNA synthesis can be by the chromosomal DNA We have used the to cells to chromosomal DNA A.Y. Zhang Biochem. 1995; PubMed Scopus Google Scholar). In we DNA synthesis by the in cells with different Lys114 The were synchronized with and cells were at the non-permissive used to the chromosomal DNA with wild-type CDC6 and used as the and The DNA and and for the as In the K114E transformant and K114P transformant a chromosomal chromosomal DNA in cells with and a wild-type of at the and we that the K114E and K114P loss-of-function and partially functional proteins, and functions to the functional mutant a in the conserved ATP-binding site, we that a functional ATP-binding site in CDC6 is required for DNA synthesis in vivo. It has been that the Cdc6 is essential for and of complexes and the loading of at G1 on chromatin is the initiation of DNA replication the of we further the that the of ATP-binding of Cdc6 to the of the We the mutant cdc6(K114E) for these showed the effect on the CDC6 gene To do this, we wild-type CDC6 and mutant cdc6(K114E) genes with its on the single In the chromosomal CDC6 gene is the control of In the of in the the chromosomal wild-type CDC6 gene is to the cell In the presence of the chromosomal wild-type CDC6 gene is and Cdc6 protein is in a mutant In the cdc6(K114E) the wild-type protein is by the mutant The function of the mutant can be We have the temporal and spatial regulation of the Cdc6 nucleotide-binding as in We the association of the cdc6(K114E) mutant protein with chromatin and the loading of Mcm5 onto chromatin in prepared from the yeast cell from at the points and with the association of Cdc6 with or with Mcm5 associated with In the presence of the Cdc6 growth of the cell on Cdc6 from the in the The were synchronized by the on the of cells and the of the S is in the wild-type cells. which used as control showed a throughout the cell cycle not The association of wild-type Cdc6 with chromatin in a cell cycle-dependent Cdc6 when the cell from G1 It on chromatin G1 at the of S and from chromatin and and The association of Mcm5 with chromatin showed a to that of the Cdc6 Mcm5 onto chromatin G1 from chromatin S and not and The association of cdc6(K114E) mutant with different from that the wild-type Cdc6 The cdc6(K114E) can with chromatin but from the the same the association of Mcm5 with chromatin in the presence of the mutant cdc6(K114E) proteins. The loading of the Mcm5 protein onto chromatin for at the cell from the G1 or for Cdc6 and suggesting that the cell cycle and the cell to with these in the mutant is to defective DNA synthesis the the chromatin and the of the cell cycle progression in the cdc6(K114E) the of Cdc6 we the of Orc1 with In the Orc1 and Cdc6 in a protein complex. and A. to their has been that the Cdc6 and Orc1 may be in a single gene family J. J. 1995; Full Text PDF PubMed Scopus Google Scholar). This to investigate the between Cdc6 and Orc1 in vitro and in vivo. In the in vitro interaction protein on the used as a protein The same the Orc1 used as Yeast as the Cdc6 protein were the the were by protein The presence of Cdc6 protein can be by interaction between Orc1 and Cdc6 To test is a direct interaction between Cdc6 and we used wild-type and mutant for interaction In the that yeast can be In the wild-type to with suggesting a direct Intriguingly, This that Orc1 not with cdc6(K114E) To we used mutant protein as The of the protein has been as to Glu This leads to a temperature-sensitive mutation. is defect in interaction The of interaction is not to the of as the same of cdc6(K114E) and were and used in In in vivo interaction protein in This wild-type mutant or proteins, The protein isolated by and with In is between Orc1 and Cdc6 but the interaction is in of Orc1 and cdc6(K114E) mutant protein interaction between Orc1 and temperature-sensitive protein suggesting that the a defective mechanism different from the in ATP-binding mutation. the results suggest that may or a proper of Cdc6, which is for Orc1 The of the proper interaction may to the of replicative complex Cdc6 essential role in the initiation of DNA replication. is known in eukaryotic Cdc6 contains a conserved known as Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). This may a role in the of The Lys is essential for interaction between the and the show that the in yeast Cdc6 with the for ATP-binding protein not In order to the conserved Lys essential amino acid as in other nucleotide-binding proteins, we have the Lys to five other amino In with the nucleotide-binding site sequence the D. S. EMBO J. PubMed Scopus Google Scholar). In other and proteins, sequence has been to functional Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). It has been demonstrated that the can and in It has been that Cdc6 is as W. Rottjakob H.-W. Kuntzel H. J. Biol. Chem. 1994; 269: 23351-23356Abstract Full Text PDF PubMed Google Scholar). The in vivo of in Cdc6 is of the conserved Lys may be to its vivo. Indeed, we five at Lys114 of the nucleotide-binding We have used to the effect on the cell functions of various of complementation of the temperature-sensitive effect on the growth of plasmid stability in the transformant and of DNA synthesis in these The to DNA synthesis in and transformant cells synthesis on chromosomal DNA is may be that the among for yeast chromosomal DNA synthesis. Our demonstrated that a functional is required for the CDC6 in DNA synthesis. Our results that the of Lys to Glu (K114E) and to Pro (K114P) to loss-of-function in of these of the Lys to Gln (K114Q) and to Leu (K114L) leads to partially functional proteins. a equivalent to functional to contains Arg in of Lys at the same A.E. Res. PubMed Scopus Google Scholar), that may not be a function of in This leads to the that may be in or in the in events at chromatin in the G1/S transition of the cell cycle. do these with the in of initiation of DNA The Cdc6 protein is required for and of and the Cdc6 protein is required for the loading of proteins. In the Cdc6 protein with protein complex is at late S. Jong A. Biol. 1996; PubMed Scopus Google Scholar). The protein kinase may activate the initiation of DNA replication E. Nasmyth K. 1994; Full Text PDF PubMed Scopus Google Scholar). It is that a of the functional replication complex is required for the of the DNA replication and cell cycle control 1996; PubMed Scopus Google Scholar). It is that is essential for DNA but is in eukaryotic DNA replication. We have the temporal and spatial regulation of the wild-type CDC6 and cdc6(K114E) gene to the of in This is and for the molecular events from late G1 the S DNA Indeed, we demonstrated that the function of cdc6(K114E) leads to of the replicative complex. At the of replicative complexes from a to the molecular mechanism of we have demonstrated that is a direct interaction between yeast Orc1 and Cdc6 proteins. The is with from other the and functions of the and the interaction between Cdc6 and Orc1 is conserved in eukaryotes Chen J. M. A. Biol. PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar). we demonstrated that the interaction between Orc1 and Cdc6 is defective in the cdc6(K114E) mutation. This is The is that the ATP-binding to complex which results in a in the of operational complex. the requirement of for interaction is in a of DNA replication. This is by chromatin studies In with the of these we have demonstrated that the in vivo function of the conserved Lys114 in the nucleotide-binding site of CDC6 is required for cell growth and DNA synthesis. We have demonstrated at the molecular the mutant at its to with The that the ATP-binding is highly conserved among Cdc6 and Orc1 protein their role in the initiation of DNA replication Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, E. Nasmyth K. 1994; Full Text PDF PubMed Scopus Google Scholar). by or to the functions in the events initiation of replication Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Google Scholar, J. J. 1995; Full Text PDF PubMed Scopus Google Scholar). The highly conserved central of the and family contains a for M. 1995; PubMed Scopus Google Scholar). It is to that the of the by the Cdc6 and Orc1 or to the sequence initiation of replication. in the and may a studies of the roles of in and may regulation in DNA replication and cell cycle We Chen for with the