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Drosophila Mcm10 is required for DNA replication and differentiation in the compound eye.

ABSTRACT: Mini chromosome maintenance 10 (Mcm10) is an essential protein, which is conserved from S. cerevisiae to Drosophila and human, and is required for the initiation of DNA replication. Knockdown of Drosophila Mcm10 (dMcm10) by RNA interference in eye imaginal discs induces abnormal eye morphology (rough eye phenotype), and the number of ommatidia is decreased in adult eyes. We also observed a delay in the S phase and M phase in eye discs of dMcm10 knockdown fly lines. These results show important roles for dMcm10 in the progression of S and M phases. Furthermore, genome damage and apoptosis were induced by dMcm10 knockdown in eye imaginal discs. Surprisingly, when we used deadpan-lacZ and klingon-lacZ enhancer trap lines to monitor the photoreceptor cells in eye discs, knockdown of dMcm10 by the GMR-GAL4 driver reduced the signals of R7 photoreceptor cells. These data suggest an involvement of dMcm10 in R7 cell differentiation. This involvement appears to be independent of the apoptosis induced by dMcm10 knockdown. Together, these results suggest that dMcm10 knockdown has an effect on DNA replication and R7 cell differentiation.

SUBMITTER: Vo N

PROVIDER: S-EPMC3970972 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Novel roles of HP1a and Mcm10 in DNA replication, genome maintenance and photoreceptor cell differentiation.

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Project description:The eukaryotic DNA replication initiation factor Mcm10 is essential for both replisome assembly and function. Human Mcm10 has two DNA-binding domains, the conserved internal domain (ID) and the C-terminal domain (CTD), which is specific to metazoans. SIRT1 is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that belongs to the sirtuin family. It is conserved from yeast to human and participates in cellular controls of metabolism, longevity, gene expression and genomic stability. Here we report that human Mcm10 is an acetylated protein regulated by SIRT1, which binds and deacetylates Mcm10 both in vivo and in vitro, and modulates Mcm10 stability and ability to bind DNA. Mcm10 and SIRT1 appear to act synergistically for DNA replication fork initiation. Furthermore, we show that the two DNA-binding domains of Mcm10 are modulated in distinct fashion by acetylation/deacetylation, suggesting an integrated regulation mechanism. Overall, our study highlights the importance of protein acetylation for DNA replication initiation and progression, and suggests that SIRT1 may mediate a crosstalk between cellular circuits controlling metabolism and DNA synthesis.

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Project description:Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication.

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