Abstract
Replication of genetic information is one of the most important biological processes for survival. DNA helicases are important initiating molecular motors, which are required for the unwinding of nucleic acids. In this study we are looking at the Fanconi Anemia group J (FANCJ) protein. FANCJ is a superfamily 2 helicase and has roles that span from unwinding complex DNA substrates, to cancer suppression, and inter-strand cross-link repair. One of the main aims of this study is to characterize the [Fe-S] cluster of FANCJ. Towards this goal, a truncated version of FANCJ (FANCJ-Tr1) was engineered, containing the N-terminal [Fe-S] cluster. Two different solubility enhancement tags (SETs) were employed, pSUMO and MBP. Initially, the truncated protein underwent attempts at isolation under native and nonnative conditions. The native preparations were deemed unsuccessful due to undesirable biophysical behavior, while the non-native preparations were successful in isolating the protein, but refolding was unsuccessful. Isolation of the MBP-FANCJ-Tr1 was successful and allowed purification of the protein containing the [Fe-S] cluster. This is the first time the presence and chemical nature of the [Fe-S] cluster in FANCJ has been demonstrated and sets the stage for future studies to assess the role of the [Fe-S] cluster in the protein (mal)function and disease propagation.