Generation of a novel endogenously StrepII/FLAG tagged system for the identification of the vertebrate FANCJ associated proteins

Abstract

Fanconi Anaemia (FA) is a rare inherited chromosomal instability disorder characterized by developmental abnormalities, bone marrow failure, increased risk of developing cancer and an increased cellular sensitivity to DNA interstrand crosslinking compounds, including the anticancer drugs, cisplatin, nitrogen mustard and mitomycin C. From a therapeutic point of view, it is important to understand how cells respond to and repair DNA damage caused by crosslinking compounds, used to treat cancer. To date, fifteen different proteins involved in a common pathway, known as the FA pathway have been identified and are known to respond to, and influence repair of interstrand crosslinks (ICLs). So far, the role of these proteins in ICL repair remains elusive. To gain insights into the molecular basis of ICL repair, we chose to study the FA-associated helicase FANCJ, as it is known to bind and metabolize a variety of DNA substrates, implicating it in maintenance of genomic stability. Here, I report a novel genetic-proteomic approach to study FANCJ. This system uses epitope tagged FANCJ expressed from its chromosomal locus, in chicken DT40 cells, kept under the control of its endogenous promoter. Tandem affinity purification is employed to purify and isolate the tagged protein and identify interacting partners of interest. To date, I have generated, characterized and validated epitope tagged FANCJ cell lines. Pilot immunoprecipitations were carried out to establish the efficiency and reproducibility of the immunoprecipitations (IPs). Analysis by mass spectrometry revealed the presence of epitope tagged FANCJ in scaled-up immunoprecipitations. In the future, these cell lines will be used to identify and characterize the FANCJ interactome.This thesis is not currently available via ORA