Drosophila Mcm10 in DNA Replication and Heterochromatin Dynamics

Cornell Univ - State: Awds Made Prior May 2010, Ithaca NY

Investigators

Abstract

The chromosome cycle begins with the precise replication of the genome, followed by condensation of the duplicated genomes and segregation of sister chromatids. These processes define the distinct phases of a cell division cycle. Defects in, or mis-coordination of these processes can have dire consequences to a proliferating cell resulting in chromosome damage, genome instability and chromosome mis-segregation. The possible roles of the Drosophila MCM10 protein in DNA replication and heterochromatin formation and its effects on variegated gene expression, maintenance of genome integrity and chromosome segregation will be explored in this project. MCM10 was initially characterized in the budding and fission yeast as an abundant nuclear protein that plays an important role in the initiation of DNA replication. MCM10 is localized to replication origins, interacts physically and functionally with subunits of the presumptive replicative MCM helicase and facilitates the phosphorylation of the MCM helicase by the Cdc7-Dbf4 kinase, a step believed to activate the MCM helicase. Studies in Xenopus laevis confirmed the role of MCM10 in the initiation step and suggest that MCM10 plays a critical role in bridging the activation of the MCM helicase with the recruitment of factors required for DNA synthesis. Recent Drosophila studies by the Tye laboratory showed that MCM10 interacts not only with components of the pre-replication complex that is assembled at replication origins during G1 phase, it also interacts with components of the elongation machinery. Furthermore, it interacts with components of the heterochromatin complex including ORC and HP1. RNA interference studies in tissue culture cells indicate that depletion of MCM10 leads to abnormal chromosome morphology such as aberrant chromosome condensation, precocious sister chromatid disjunction and possibly chromosome fragmentation. These chromosomal abnormalities may be the indirect result of defects in DNA replication that affect chromatin condensation. Alternatively, MCM10 may play a direct role in both DNA replication and chromatin condensation. MCM10's role in DNA replication and the maintenance of chromosome integrity in Drosophila will be explored at the cellular level by addressing whether MCM10 depletion affects DNA replication and whether MCM10 depletion affects heterochromatin formation. The developmental abnormalities that result from depletion of MCM10 will also be investigated at the organismal level. The approaches taken will include construction of genetic null mutants, hypomorphic mutants and conditional transgenic RNAi mutants, mosaic analysis, PEV analysis, chromosome cytology, CFP and YFP colocalization as well as FRET analysis, and two-hybrid analysis. Understanding biology requires understanding chromosomal biology- how the integrity of genetic information is preserved through the precise replication and segregation of chromosomes and how that information is selectively disseminated throughout development. This project should increase the depth and breadth of our understanding of the mechanisms through which chromosomes carry out their many functions. In addition, this research will help train undergraduates and a graduate student and help a new investigator develop his teaching skills.