The second main area of the research focus of my lab is to define the role of the replication initiator protein Orc6 in cytokinesis and its interaction with proteins that govern the chromosome and cell division cycle in human cells.
Role of Orc6 proteins in cell-cycle progression and cytokinesis. Also, structure function analysis of ORC and the heterochromatin interacting partners would provide better insights into the understanding of ORCs involvement in heterochromatin organization and chromosome structure. Further, live cell imaging using mitotic markers will enable to study the order of the mitotic defects in ORC depleted cells. Studying the localization of proteins which interact with ORC at heterochromatin with respect to differential levels of ORC proteins as well as dissecting the functional significance of their interaction will be critical towards understanding how ORC is involved in organizing chromatin structure during different stages of the cell-cycle. How the Orc2 depletion results in the abnormal localization of HP1 proteins from mitotic chromosomes remains to be addressed. In Orc2 depleted cells, a population of cells arrests in mitosis with defective chromosome structure and condensation, spindle and chromosome congression defects. The loss of Orc2 and Orc3 proteins from human cells also results in the loss of HP1 (HP1a and b) from the centric heterochromatin during mitosis. In the fission yeast Schizosaccharomyces pombe, the homologue of the heterochromatin protein (HP1) protein Swi6 is critical for efficient sister chromatid cohesion during cell division. It has been suggested that centromeric heterochromatin is necessary for both the cohesion of sister chromatids and the subsequent normal disjunction of mitotic chromosomes. To further pinpoint the role of ORC subunits at heterochromatin, my lab focuses on the identification of the interacting partners of ORCs at heterochromatin during different stages of the cell cycle and to delineate the replication function of ORC proteins versus their role in chromatin compaction. Role of ORC proteins in heterochromatin organization and chromosome structure: ORC proteins have been implicated in heterochromatin organization in mammalian cells (Figure 1) and Drosophila and gene silencing in Saccharomyces cerevisiae. The focal point of research in my lab is to study the events and uncover the cues that integrate DNA replication with heterochromatin organization, chromosome segregation and cytokinesis, major focus being on the role of ORC in interconnecting these events.
Other than its bonafide role in DNA replication, ORC proteins are involved in diverse functions including gene silencing, heterochromatin organization, cytokinesis and also in dendrite formation in postmitotic neurons. Thus the accurate duplication of DNA is of paramount importance and is governed by a number of proteins including the Origin Recognition complex (ORC) which serves as a landing pad for the assembly of a multiprotein pre-replicative complex. Inaccurate DNA replication in turns leads to abnormal chromosome segregation resulting in aneuploidy and genomic instability, a hallmark of most cancerous cells. DNA replication, which occurs during S phase of the cell cycle, is intimately linked to mitotic progression and eventually cell division. The initiation of DNA replication in eukaryotic cells is a highly regulated process that leads to the duplication of genetic information for the next cell generation.