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Mcm10 (Dna43), first identified in Saccharomyces cerevisiae, is an essential protein which functions in the initiation of DNA synthesis. Mcm10 is a nuclear protein that is localized to replication origins and mediates the interaction of the Mcm2-7 complex with replication origins. We identified and cloned a human cDNA whose product was structurally homologous to the yeast Mcm10 protein. Human Mcm10 (HsMcm10) is a 98-kDa protein of 874 amino acids which shows 23 and 21% overall similarity to Schizosaccharomyces pombe Cdc23 and S. cerevisiae Mcm10, respectively. The messenger RNA level of HsMcm10 increased at the G(1)/S-boundary when quiescent human NB1-RGB cells were induced to proliferate as is the case of many replication factors. HsMcm10 associated with nuclease-resistant nuclear structures throughout S phase and dissociated from it in G(2) phase. HsMcm10 associated with human Orc2 protein when overexpressed in COS-1 cells. HsMcm10 also interacted with Orc2, Mcm2 and Mcm6 proteins in the yeast two-hybrid system. These results suggest that HsMcm10 may function in DNA replication through the interaction with Orc and Mcm2-7 complexes.

The prevalence of BRCA1/2 germline mutations in Japanese patients suspected to have hereditary breast/ovarian cancer was examined by a multi-institutional study, aiming at the clinical application of total sequencing analysis and validation of assay sensitivity in Japanese people using a cross-sectional approach based on genetic factors estimated from personal and family histories. One hundred and thirty-five subjects were referred to the genetic counseling clinics and enrolled in the study. Full sequencing analysis of the BRCA1/2 gene showed 28 types of deleterious mutations in 36 subjects (26.7%), including 13 types of BRCA1 mutations in 17 subjects (12.6%) and 15 types of BRCA2 mutations in 19 subjects (14.1%). Subjects were classified into five groups and 22 subgroups according to their personal and family history of breast and/or ovarian cancer, and the prevalence of deleterious mutations was compared with previously reported data in non-Ashkenazi individuals. Statistical analysis using the Mantel-Haenszel test for groups I through IV revealed that the prevalence of Japanese subjects was significantly higher than that of non-Ashkenazi individuals (P = 0.005, odds ratio 1.87, 95% confidence interval 1.22-2.88). Family history of the probands suffering from breast cancer indicated risk factors for the presence of deleterious mutations of BRCA1/2 as follows: (1) I n Japan, breast cancer is the most frequent malignancy in women and estimates of new cases and deaths in 2002 were 32 245 and 9178, respectively.(1) The standardized incidence ratio of breast cancer in Japan was approximately one-third that of the US (32.7 vs 101.7 per 100 000 women).(1) The incidence of breast cancer in Japanese women shows a steady increase; however, it is still much lower than in Western countries. In breast cancer, family history is the strongest risk factor for cancer predisposition. Epidemiological studies showed that 12% of women with breast cancer have one affected family member and 1% have two or more affected relatives.(2) Women with one, two, and three or more first-degree affected relatives have an increased breast cancer risk when compared with women who do not have an affected relative (risk ratios 1.8, 2.9, and 3.9, respectively).(2) Recent advances in molecular genetics elucidated BRCA1 and BRCA2 (BRCA1/2) as two major susceptibility genes for breast cancer predisposition.(3,4) Gene testing of BRCA1/2 is available as a routine clinical test for diagnosing hereditary breast/ovarian cancer (HBOC) in the US and other Western countries, (5,6) while only a few reports have been published concerning the prevalence of BRCA1/2 mutations among Japanese people. (7)(8)(9)(10)(11)(12) The methods of genetic analysis employed in these studies varied, such as polymerase chain reaction (PCR)/ single strand conformational polymorphisms (SSCP), protein truncation test, and PCR/direct sequencing, but they were performed as preliminary in-house tests in the research setting. In the US, commercial BRCA1/2 gene testing was initiated by M...

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A-and B-type lamins are integrated into the lamina.

In eukaryotes, initiation of DNA replication is a strictly controlled process, so that chromosomal DNA is precisely duplicated once per cell cycle. Recent studies using different systems show that a number of proteins are involved in the initiation of DNA replication, a process that is largely conserved from yeast to human (1, 2). Current models indicate that initiation consists of two steps. In the first step, the origin recognition complex (ORC), 1 Cdc6, Cdt1, and Mcm2-7 proteins sequentially assemble on the replication origins to form the prereplicative complex, from late mitosis to early G 1 phase. The ORC, a complex of six proteins (Orc1-6), binds replication origins (3, 4) and recruits Cdc6. Cdc6 in turn coordinates with Cdt1 to load the Mcm2-7 complex (5-10), a presumed replicative helicase (11, 12), on the chromatin template. In the second step, Cdc7/Dbf4 kinase and S phase cyclin-dependent kinases (S-Cdks) activate the prereplicative complex and trigger DNA replication by loading Cdc45 onto each origin with programmed timing (13-16). Cdc45 facilitates assembly of the replication machinery by recruiting replication protein A and DNA polymerases (17, 18).Eukaryotes contain multiple parallel pathways to ensure that the prereplicative complex is not re-assembled until the segregation of chromosomes in mitosis. Cdc6 is either degraded via the ubiquitin-dependent pathway (19) or exported out of the nucleus (20). Phosphorylation of Mcm2-7 complex by cdc2 kinase initiates dissociation from chromatin during S phase (21). Cdt1 is regulated by protein expression as well as interactions with geminin to ensure that it is active only in the G 1 phase (22,23).Mcm10 (Dna43) was originally discovered in Saccharomyces cerevisiae while screening to identify other mcm mutants (24). Previous studies performed in S. cerevisiae suggest that Mcm10 has multiple roles in DNA replication. The mcm10 mutant is defective in initiation of DNA replication at the non-permissive temperature (24) and causes stalling of replication forks when the replication machinery passes through origins that do not fire (24). In addition, Mcm10 mediates the loading of the Mcm2-7 complex onto replication origins (25), and interacts genetically with Cdc45, DNA polymerase ␦ and ⑀, which are required for the elongation steps of DNA replication (26). Therefore, it appears that Mcm10 is involved in both origin activation and elongation, although the mechanisms by which the protein interacts with multiple replication factors at each step remain to be elucidated.Mcm10 homologs have additionally been discovered in Schizosaccharomyces pombe and Caenorhabditis elegans (25,27). Recently, we identified Drosophila and human homologs of Mcm10 and demonstrated that human Mcm10 interacts with the mammalian Orc2 and Mcm2-7 complex (28). We also confirmed that human Mcm10 binds chromatin during S phase and dissociates in G 2 phase (28), whereas yeast Mcm10 remains bound to chromatin throughout the cell cycle (25,26). To clarify the mechanism of regulation of Mcm10 funct...

Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian cells by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bsr had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for mammalian cells.

Chromosomal double-strand breaks (DSBs) in mammalian cells are usually repaired through either of two pathways: end-joining (EJ) or homologous recombination (HR). To clarify the relative contribution of each pathway and the ensuing genetic changes, we developed a system to trace the fate of DSBs that occur in an endogenous single-copy human gene. Lymphoblastoid cell lines TSCE5 and TSCER2 are heterozygous (+/-) or compound heterozygous (-/-), respectively, for the thymidine kinase gene (TK), and we introduced an I-SceI endonuclease site into the gene. EJ for a DSB at the I-SceI site results in TK-deficient mutants in TSCE5 cells, while HR between the alleles produces TK-proficient revertants in TSCER2 cells. We found that almost all DSBs were repaired by EJ and that HR rarely contributes to the repair in this system. EJ contributed to the repair of DSBs 270 times more frequently than HR. Molecular analysis of the TK gene showed that EJ mainly causes small deletions limited to the TK gene. Seventy percent of the small deletion mutants analyzed showed 100- to 4,000-bp deletions with a 0- to 6-bp homology at the joint. Another 30%, however, were accompanied by complicated DNA rearrangements, presumably the result of sister-chromatid fusion. HR, on the other hand, always resulted in non-crossing-over gene conversion without any loss of genetic information. Thus, although HR is important to the maintenance of genomic stability in DNA containing DSBs, almost all chromosomal DSBs in human cells are repaired by EJ.

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