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. 1997 Nov;147(3):1025–1041. doi: 10.1093/genetics/147.3.1025

Mutational Analysis of Cdc19p, a Schizosaccharomyces Pombe Mcm Protein

S L Forsburg 1, D A Sherman 1, S Ottilie 1, J R Yasuda 1, J A Hodson 1
PMCID: PMC1208231  PMID: 9383050

Abstract

The cdc19(+) gene encodes an essential member of the MCM family of replication proteins in Schizosaccharomyces pombe. We have examined the structure and function of the Cdc19p protein using molecular and genetic approaches. We find that overproduction of wild-type Cdc19p in wild-type cells has no effect, but cdc19-P1 mutant cells do not tolerate elevated levels of other MCM proteins or overexpression of mutant forms of Cdc19p. We have found genetic interactions between cdc19(+) and genes encoding subunits of DNA polymerase {delta small} and the replication initiator cdc18(+). We have constructed a series of point mutations and sequence deletions throughout Cdc19p, which allow us to distinguish essential from nonessential regions of the protein. Not surprisingly, conserved residues in the MCM homology domain are required for protein function, but some residues outside the core homology domain are dispensable.

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Selected References

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  1. Basi G., Schmid E., Maundrell K. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene. 1993 Jan 15;123(1):131–136. doi: 10.1016/0378-1119(93)90552-e. [DOI] [PubMed] [Google Scholar]
  2. Burkhart R., Schulte D., Hu D., Musahl C., Göhring F., Knippers R. Interactions of human nuclear proteins P1Mcm3 and P1Cdc46. Eur J Biochem. 1995 Mar 1;228(2):431–438. [PubMed] [Google Scholar]
  3. Carpenter P. B., Mueller P. R., Dunphy W. G. Role for a Xenopus Orc2-related protein in controlling DNA replication. Nature. 1996 Jan 25;379(6563):357–360. doi: 10.1038/379357a0. [DOI] [PubMed] [Google Scholar]
  4. Chong J. P., Mahbubani H. M., Khoo C. Y., Blow J. J. Purification of an MCM-containing complex as a component of the DNA replication licensing system. Nature. 1995 Jun 1;375(6530):418–421. doi: 10.1038/375418a0. [DOI] [PubMed] [Google Scholar]
  5. Fantes P. Epistatic gene interactions in the control of division in fission yeast. Nature. 1979 May 31;279(5712):428–430. doi: 10.1038/279428a0. [DOI] [PubMed] [Google Scholar]
  6. Fenech M., Carr A. M., Murray J., Watts F. Z., Lehmann A. R. Cloning and characterization of the rad4 gene of Schizosaccharomyces pombe; a gene showing short regions of sequence similarity to the human XRCC1 gene. Nucleic Acids Res. 1991 Dec 25;19(24):6737–6741. doi: 10.1093/nar/19.24.6737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fernandez Sarabia M. J., McInerny C., Harris P., Gordon C., Fantes P. The cell cycle genes cdc22+ and suc22+ of the fission yeast Schizosaccharomyces pombe encode the large and small subunits of ribonucleotide reductase. Mol Gen Genet. 1993 Apr;238(1-2):241–251. doi: 10.1007/BF00279553. [DOI] [PubMed] [Google Scholar]
  8. Forsburg S. L. Comparison of Schizosaccharomyces pombe expression systems. Nucleic Acids Res. 1993 Jun 25;21(12):2955–2956. doi: 10.1093/nar/21.12.2955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Francesconi S., Park H., Wang T. S. Fission yeast with DNA polymerase delta temperature-sensitive alleles exhibits cell division cycle phenotype. Nucleic Acids Res. 1993 Aug 11;21(16):3821–3828. doi: 10.1093/nar/21.16.3821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hennessy K. M., Lee A., Chen E., Botstein D. A group of interacting yeast DNA replication genes. Genes Dev. 1991 Jun;5(6):958–969. doi: 10.1101/gad.5.6.958. [DOI] [PubMed] [Google Scholar]
  11. Hirano T., Hiraoka Y., Yanagida M. A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J Cell Biol. 1988 Apr;106(4):1171–1183. doi: 10.1083/jcb.106.4.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Iino Y., Yamamoto M. The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase delta. Mol Gen Genet. 1997 Mar 18;254(1):93–97. doi: 10.1007/s004380050395. [DOI] [PubMed] [Google Scholar]
  13. Ishimi Y., Ichinose S., Omori A., Sato K., Kimura H. Binding of human minichromosome maintenance proteins with histone H3. J Biol Chem. 1996 Sep 27;271(39):24115–24122. doi: 10.1074/jbc.271.39.24115. [DOI] [PubMed] [Google Scholar]
  14. Kearsey S. E., Maiorano D., Holmes E. C., Todorov I. T. The role of MCM proteins in the cell cycle control of genome duplication. Bioessays. 1996 Mar;18(3):183–190. doi: 10.1002/bies.950180305. [DOI] [PubMed] [Google Scholar]
  15. Kelly T. J., Martin G. S., Forsburg S. L., Stephen R. J., Russo A., Nurse P. The fission yeast cdc18+ gene product couples S phase to START and mitosis. Cell. 1993 Jul 30;74(2):371–382. doi: 10.1016/0092-8674(93)90427-r. [DOI] [PubMed] [Google Scholar]
  16. Koonin E. V. A common set of conserved motifs in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initiation of eukaryotic DNA replication. Nucleic Acids Res. 1993 Jun 11;21(11):2541–2547. doi: 10.1093/nar/21.11.2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Krause T., Kunau W. H., Erdmann R. Effect of site-directed mutagenesis of conserved lysine residues upon Pas1 protein function in peroxisome biogenesis. Yeast. 1994 Dec;10(12):1613–1620. doi: 10.1002/yea.320101210. [DOI] [PubMed] [Google Scholar]
  18. Kroll E. S., Hyland K. M., Hieter P., Li J. J. Establishing genetic interactions by a synthetic dosage lethality phenotype. Genetics. 1996 May;143(1):95–102. doi: 10.1093/genetics/143.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Krude T., Musahl C., Laskey R. A., Knippers R. Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication. J Cell Sci. 1996 Feb;109(Pt 2):309–318. doi: 10.1242/jcs.109.2.309. [DOI] [PubMed] [Google Scholar]
  20. Lanford R. E., Butel J. S. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell. 1984 Jul;37(3):801–813. doi: 10.1016/0092-8674(84)90415-x. [DOI] [PubMed] [Google Scholar]
  21. Laurent B. C., Yang X., Carlson M. An essential Saccharomyces cerevisiae gene homologous to SNF2 encodes a helicase-related protein in a new family. Mol Cell Biol. 1992 Apr;12(4):1893–1902. doi: 10.1128/mcb.12.4.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lei M., Kawasaki Y., Tye B. K. Physical interactions among Mcm proteins and effects of Mcm dosage on DNA replication in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Sep;16(9):5081–5090. doi: 10.1128/mcb.16.9.5081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li J. J., Herskowitz I. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science. 1993 Dec 17;262(5141):1870–1874. doi: 10.1126/science.8266075. [DOI] [PubMed] [Google Scholar]
  24. Lowndes N. F., McInerny C. J., Johnson A. L., Fantes P. A., Johnston L. H. Control of DNA synthesis genes in fission yeast by the cell-cycle gene cdc10+. Nature. 1992 Jan 30;355(6359):449–453. doi: 10.1038/355449a0. [DOI] [PubMed] [Google Scholar]
  25. Ma L., Westbroek A., Jochemsen A. G., Weeda G., Bosch A., Bootsma D., Hoeijmakers J. H., van der Eb A. J. Mutational analysis of ERCC3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function. Mol Cell Biol. 1994 Jun;14(6):4126–4134. doi: 10.1128/mcb.14.6.4126. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Madine M. A., Khoo C. Y., Mills A. D., Musahl C., Laskey R. A. The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts. Curr Biol. 1995 Nov 1;5(11):1270–1279. doi: 10.1016/s0960-9822(95)00253-3. [DOI] [PubMed] [Google Scholar]
  27. Maiorano D., Van Assendelft G. B., Kearsey S. E. Fission yeast cdc21, a member of the MCM protein family, is required for onset of S phase and is located in the nucleus throughout the cell cycle. EMBO J. 1996 Feb 15;15(4):861–872. [PMC free article] [PubMed] [Google Scholar]
  28. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  29. Maundrell K. nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine. J Biol Chem. 1990 Jul 5;265(19):10857–10864. [PubMed] [Google Scholar]
  30. Miyake S., Okishio N., Samejima I., Hiraoka Y., Toda T., Saitoh I., Yanagida M. Fission yeast genes nda1+ and nda4+, mutations of which lead to S-phase block, chromatin alteration and Ca2+ suppression, are members of the CDC46/MCM2 family. Mol Biol Cell. 1993 Oct;4(10):1003–1015. doi: 10.1091/mbc.4.10.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Miyake S., Saito I., Kobayashi H., Yamashita S. Identification of two Xenopus laevis genes, xMCM2 and xCDC46, with sequence homology to MCM genes involved in DNA replication. Gene. 1996 Oct 10;175(1-2):71–75. doi: 10.1016/0378-1119(96)00122-9. [DOI] [PubMed] [Google Scholar]
  32. Musahl C., Schulte D., Burkhart R., Knippers R. A human homologue of the yeast replication protein Cdc21. Interactions with other Mcm proteins. Eur J Biochem. 1995 Jun 15;230(3):1096–1101. doi: 10.1111/j.1432-1033.1995.tb20660.x. [DOI] [PubMed] [Google Scholar]
  33. Muzi Falconi M., Brown G. W., Kelly T. J. cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1566–1570. doi: 10.1073/pnas.93.4.1566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nasmyth K., Nurse P. Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet. 1981;182(1):119–124. doi: 10.1007/BF00422777. [DOI] [PubMed] [Google Scholar]
  35. Okishio N., Adachi Y., Yanagida M. Fission yeast Nda1 and Nda4, MCM homologs required for DNA replication, are constitutive nuclear proteins. J Cell Sci. 1996 Feb;109(Pt 2):319–326. doi: 10.1242/jcs.109.2.319. [DOI] [PubMed] [Google Scholar]
  36. Pignède G., Bouvier D., de Recondo A. M., Baldacci G. Characterization of the POL3 gene product from Schizosaccharomyces pombe indicates inter-species conservation of the catalytic subunit of DNA polymerase delta. J Mol Biol. 1991 Nov 20;222(2):209–218. doi: 10.1016/0022-2836(91)90207-m. [DOI] [PubMed] [Google Scholar]
  37. Romanowski P., Madine M. A., Rowles A., Blow J. J., Laskey R. A. The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin. Curr Biol. 1996 Nov 1;6(11):1416–1425. doi: 10.1016/s0960-9822(96)00746-4. [DOI] [PubMed] [Google Scholar]
  38. Rowles A., Chong J. P., Brown L., Howell M., Evan G. I., Blow J. J. Interaction between the origin recognition complex and the replication licensing system in Xenopus. Cell. 1996 Oct 18;87(2):287–296. doi: 10.1016/s0092-8674(00)81346-x. [DOI] [PubMed] [Google Scholar]
  39. Russell P., Nurse P. cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell. 1986 Apr 11;45(1):145–153. doi: 10.1016/0092-8674(86)90546-5. [DOI] [PubMed] [Google Scholar]
  40. Saka Y., Yanagida M. Fission yeast cut5+, required for S phase onset and M phase restraint, is identical to the radiation-damage repair gene rad4+. Cell. 1993 Jul 30;74(2):383–393. doi: 10.1016/0092-8674(93)90428-s. [DOI] [PubMed] [Google Scholar]
  41. Sazer S., Sherwood S. W. Mitochondrial growth and DNA synthesis occur in the absence of nuclear DNA replication in fission yeast. J Cell Sci. 1990 Nov;97(Pt 3):509–516. doi: 10.1242/jcs.97.3.509. [DOI] [PubMed] [Google Scholar]
  42. Schulte D., Richter A., Burkhart R., Musahl C., Knippers R. Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm proteins. Eur J Biochem. 1996 Jan 15;235(1-2):144–151. doi: 10.1111/j.1432-1033.1996.00144.x. [DOI] [PubMed] [Google Scholar]
  43. Simanis V., Nurse P. The cell cycle control gene cdc2+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell. 1986 Apr 25;45(2):261–268. doi: 10.1016/0092-8674(86)90390-9. [DOI] [PubMed] [Google Scholar]
  44. Singh S. K., Maurizi M. R. Mutational analysis demonstrates different functional roles for the two ATP-binding sites in ClpAP protease from Escherichia coli. J Biol Chem. 1994 Nov 25;269(47):29537–29545. [PubMed] [Google Scholar]
  45. Sinha P., Chang V., Tye B. K. A mutant that affects the function of autonomously replicating sequences in yeast. J Mol Biol. 1986 Dec 20;192(4):805–814. doi: 10.1016/0022-2836(86)90030-6. [DOI] [PubMed] [Google Scholar]
  46. Starborg M., Brundell E., Gell K., Larsson C., White I., Daneholt B., Hög C. A murine replication protein accumulates temporarily in the heterochromatic regions of nuclei prior to initiation of DNA replication. J Cell Sci. 1995 Mar;108(Pt 3):927–934. doi: 10.1242/jcs.108.3.927. [DOI] [PubMed] [Google Scholar]
  47. Su T. T., Feger G., O'Farrell P. H. Drosophila MCM protein complexes. Mol Biol Cell. 1996 Feb;7(2):319–329. doi: 10.1091/mbc.7.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Takahashi K., Yamada H., Yanagida M. Fission yeast minichromosome loss mutants mis cause lethal aneuploidy and replication abnormality. Mol Biol Cell. 1994 Oct;5(10):1145–1158. doi: 10.1091/mbc.5.10.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Todorov I. T., Attaran A., Kearsey S. E. BM28, a human member of the MCM2-3-5 family, is displaced from chromatin during DNA replication. J Cell Biol. 1995 Jun;129(6):1433–1445. doi: 10.1083/jcb.129.6.1433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Todorov I. T., Pepperkok R., Philipova R. N., Kearsey S. E., Ansorge W., Werner D. A human nuclear protein with sequence homology to a family of early S phase proteins is required for entry into S phase and for cell division. J Cell Sci. 1994 Jan;107(Pt 1):253–265. doi: 10.1242/jcs.107.1.253. [DOI] [PubMed] [Google Scholar]
  51. Treisman J. E., Follette P. J., O'Farrell P. H., Rubin G. M. Cell proliferation and DNA replication defects in a Drosophila MCM2 mutant. Genes Dev. 1995 Jul 15;9(14):1709–1715. doi: 10.1101/gad.9.14.1709. [DOI] [PubMed] [Google Scholar]
  52. Tye B. K. The MCM2-3-5 proteins: are they replication licensing factors? Trends Cell Biol. 1994 May;4(5):160–166. doi: 10.1016/0962-8924(94)90200-3. [DOI] [PubMed] [Google Scholar]
  53. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Whitebread L. A., Dalton S. Cdc54 belongs to the Cdc46/Mcm3 family of proteins which are essential for initiation of eukaryotic DNA replication. Gene. 1995 Mar 21;155(1):113–117. doi: 10.1016/0378-1119(94)00925-i. [DOI] [PubMed] [Google Scholar]
  55. Whiteheart S. W., Rossnagel K., Buhrow S. A., Brunner M., Jaenicke R., Rothman J. E. N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion. J Cell Biol. 1994 Aug;126(4):945–954. doi: 10.1083/jcb.126.4.945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yahraus T., Braverman N., Dodt G., Kalish J. E., Morrell J. C., Moser H. W., Valle D., Gould S. J. The peroxisome biogenesis disorder group 4 gene, PXAAA1, encodes a cytoplasmic ATPase required for stability of the PTS1 receptor. EMBO J. 1996 Jun 17;15(12):2914–2923. [PMC free article] [PubMed] [Google Scholar]

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