Interaction and Functional Analysis of Drosophila Mcm10

Abstract

In eukaryotic cells, DNA replication and the subsequent packaging of DNA into specific chromatin states are essential processes for the transmission of genetic material and genomic stability which may be linked by interactions between DNA replication proteins and chromatin association proteins. The discovery of several proteins with roles in both processes is evidence for this. Mcm10 is one such protein that has shown evidence for multiple roles in DNA replication, heterochromatin formation, and chromosome condensation. The separation of the functions of Mcm10 are investigated in this study by the analysis of two mutant alleles. A hypomorphic allele of Mcm10 demonstrates that Mcm10 has a role in heterochromatic silencing, chromosome condensation, and DNA replication. The analysis of a C terminal truncation allele indicates a role in endoreplication mediated through an interaction with Mcm2. The C terminal however, does not seem to be involved in heterochromatic silencing or chromosome condensation. The second phase of this study involves the development of a novel yeast three-hybrid system. The yeast two-hybrid system is a useful tool for detecting interactions between two proteins and identifying novel protein interactions. However, one limitation to the system is that some two-protein interactions require a third protein to stabilize or facilitate the binding between the two. In this system, we present two novel vectors; pGBKTet and pHook (derived from pDela) that are Gatewaytm compatible and allow screening for the third protein. Due to its numerous protein interactions and its exceptional abundance in the eukaryotic cell with approximately 40,000 molecules per haploid yeast cell, it has been proposed that Mcm10 not only has roles in DNA replication and heterochromatin formation, but also serves as a facilitator of other protein interactions. Using this system, it was shown that Mcm10 may participate in several ternary protein complexes.