Functional Studies Of Holliday Junction Resolvase HGEN1

One of the most pronounced characteristics of tumorigenesis is genomic instability. To perserved genomic integrity cells have evolved complex surveillance and defence mechanisms, collectively termed the DNA damage response (DDR). Key to the DDR is the DNA damage checkpoints, which constantly monitor the quality of DNA and in the case of sensing a DNA lesion communicate to the cell cycle machinery in order to coordinate repair with cell cycle progression, chromatin remodeling, transcriptional activity, or if beyond repair apotosis. DNA double-strand breaks (DSBs) is one of the most servere forms of DNA damage. DSBs are caused both by endogenous and exogenous factors, even a single unrepaired DSBs can lead to cell cytotoxicity, genome instability and eventually result in cell death, transformation or aging. The human Holliday junction resolvase hGENl is involved in DNA DSBs repair. Recent studies have showed that hGENl posses resolvase activity in vitro. Additionally, multicellular eukaryotes (yeast, human and C. elegans) defective in GENl display various defects DNA repair and recombination, suggesting that GENl might be involved in other genome stability maintenance processes besides its putative function in Holiday junction resolution. In C. elegans GENl promotes DNA damage signaling independently of ATR, separable from its role in DNA repair, suppoting a broader role of hGENl in the response to DSBs. I have constructed a GST tagged hGENl prokaryote expression system, expressed and purified GST-hGENl-C terminal fusion proteins, In addition, I have generated a hGENl specific antibody, which I have used to verified endogenous and exogenous hGENl localized in the nucleus and on the centrosomes. The localization of hGENl on centrosomes requires the N terminal1-161bp. To search for hGENl interacting partners we performed pull down experiments follow by MS analysis of specific bands. I cloned the putative interacting proteins into HA-plasmids to enable further validation and characterization of the potential interactions. By using immunofluorescence microscopy I found that hGENl depletion results in abnormal cell division. Furthermore, depletion of hGENl results in aberrant cell cycle progression and replication. Taken together these data suggests an important role of hGENl in cell cycle regulation and centrosome duplication.