The School of Molecular and Cellular Biology at the University of Illinois at Urbana-Champaign

DNA helicases are ubiquitous motor proteins that use energy of ATP binding and hydrolysis to translocate directionally on the DNA lattices, transiently unwind duplex DNA or remodel protein-nucleic acid complexes.

My interest in understanding the molecular mechanism and cellular function of DNA helicases results from their importance for virtually every aspect of DNA metabolism (including DNA repair, recombination, replication and transcription), as well as from the connection between mutations in the genes encoding DNA helicases and human genome instability disorders such as cancer and aging.

The long-term goals of our research group are to understand in enzymological and molecular terms, (1) how do the unique structural features in numerous related helicases confer upon these enzyme the capacity to perform drastically different tasks by association with and unwinding of defined sets of DNA structures, and (2) how these enzymes are integrated into various macromolecular ensembles to orchestrate complex DNA processing events.

We employ a combination of biochemical and biophysical techniques to elucidate the molecular mechanisms of the  three DNA repair helicases, archaeal Rad3 (XPD), human Bach1 (BRCA1-associated C-terminal helicase 1), and Fbh1 (F-box helicase 1) from human and fission yeast.

Rad3 and Bach1 proteins belong to the Rad3 family of DNA helicases. Enzymes in this family differ from the related helicases due to the unique insertion whose structure is stabilized by an iron-sulfur cluster and is important for the genetic stability and proficient DNA repair. Rad3 helicase (and its eukaryotic homolog, XPD) is an essential player in the nucleotide excision repair, while Bach1 activity is important for the repair of two distinct types of deletarious DNA damage: double-strand DNA breaks and inter-strand cross-links in DNA.

Fbh1 helicase plays an important role in homologous recombination, an essential cellular process whose malfunction often leads to premature aging, susceptibility to cancers and other diseases normally associated with aging. Fbh1 is a caretaker helicase that acts by preventing formation of the toxic recombination intermediates produced by Rad51 recombinase, or by resolving these intermediates.

Approaches:
An important parameter determining the cellular role of the DNA repair helicases is their ability to discriminate between various putative DNA repair intermediates. These intermediates may be represented by the synthetic DNA structures or protein-nucleic acid complexes assembled on these structures. Therefore, we carry out analyses of helicase-substrate interactions, both at the qualitative level, by identifying the preferred substrates of the selected helicases, and qualitatively, by determining the energetic contributions of the structural features characteristic of the preferred DNA substrates. We employ the gel- and fluorescence-based binding and helicase assays, DNA foot-printing and the reverse foot-printing techniques. In collaboration with Taekjip Ha’s lab (UIUC Physics) we also pursue analysis of these helicases at the level of individual protein-DNA complexes.

Coupling of a helicase with other proteins acting in the same pathway may result in the new activities that are commonly overlooked in analyses of the isolated enzymes. To characterize the Fbh1 and Bach1 helicases in the context of their respective macromolecular ensembles, we reconstitute in vitro the selected steps in the DNA repair processes and analyze the effects of these helicases on the reconstituted reactions.