Identification of Protein Interactions and Post-Translational Modifications by Mass Spectrometry that Characterize Cellular Responses to DNA Damage.

Aaron Aslanian

Appointment Period: 2005-2007 / Grant Years: [21,22]

Proliferating cells are exposed to a myriad of DNA damage sources, both intrinsic and extrinsic. Although many key players in the DNA damage response have been identified, it is still not clear how cells ultimately make the repair versus death decision. The goal of this project is to distinguish the molecular mechanisms at work using mass spectrometry to identify specific protein interactions and protein modifications that might signal toward a specific DNA damage response. Mass spectrometry (MS) experiments have been performed using PCNA as a candidate because of its well-established roles in DNA replication and DNA repair. A mouse anti-PCNA monoclonal antibody was obtained and we demonstrated that it can efficiently immunoprecipitate endogenous PCNA from HeLa cells. We are now processing the experimental PCNA samples, and expect to see changes in posttranslational modifications of PCNA (e.g. phosphorylation, ubiquitination, sumoylation, acetylation) and changes in PCNA associated proteins. Other candidate proteins, such as Dbf4, the activating subunit of Cdc7, for which we made antibodies several years ago, will be studied using similar methodology.

I am also developing methods for the identification of chromatin-associated proteins in control and MMS-treated cells. For this purpose, we are using HeLa cells that have been stably infected with a retrovirus encoding a GFP-histone H2B fusion protein. GFP-histone H2B is incorporated into nucleosomes, and is commonly used in cell biological studies to monitor chromatin dynamics. We will use anti-GFP antibodies generated in our laboratory to perform a modified chromatin immunoprecipitation (ChIP) assay. We expect to identify proteins whose association with chromatin changes in response to different extents of DNA damage, the potential roles of these proteins in the repair versus death decision will be analyzed using si/shRNA technology.

Zhang YW, Brognard J, Coughlin C, You Z, Dolled-Filhart M, Aslanian A, Manning G, Abraham RT, Hunter T. The F box protein Fbx6 regulates Chk1 stability and cellular sensitivity to replication stress. Mol Cell. (2009) 35:442-53. PMID: 19716789; PMCID: PMC2736145.

Chaurushiya MS, Lilley CE, Aslanian A, Meisenhelder J, Scott DC, Landry S,Ticau S, Boutell C, Yates JR 3rd, Schulman BA, Hunter T, Weitzman MD. ViralE3Ubiquitin Ligase-Mediated Degradation of a Cellular E3: Viral Mimicry of aCellular Phosphorylation Mark Targets the RNF8 FHA Domain. Mol Cell. 2012 Apr13;46(1):79-90. Epub 2012 Mar 7. PubMed PMID: 22405594.