Single-molecule FRET을 이용한 Caenorhabditis elegans RecQ helicase protein의 특성 연구

Abstract

The RecQ helicases are conserved from bacteria to humans and play a critical role in genome stability. In humans, mutation in RecQ genes is linked with cancer predisposition and accelerated aging. The RecQ helicases have a function of a maintenance of genome stability by RecQ helicases likely involves their participation in DNA replication, recombination, and repair pathways. They share common functions by possessing signature tandem domains, referred to as the core helicase domain, RecQ C-terminal (RQC), and helicase-and-ribonuclease D-C-terminal (HRDC) domains. One of the central questions regarding RecQ helicases is the extent to which the homologs have unique or overlapping functions. This question originally raised for the human RecQ helicases is of equal importance for other organisms, such as nematodes. C. elegans also has 4 homologous of human RecQ helicase gene. The C. elegans genome encodes four RecQ-like helicases. The genes recq1, him-6, wrn-1, and recq5 are most homologous to the human RecQ helicase genes RecQ1, BLM, WRN, and RecQ5, respectively. In this thesis, the functional dynamics of Caenorhabditis elegans RecQ helicases, HIM-6 and WRN-1 were studied at a single-molecule level in real time using single-molecule FRET technique. First, we investigated the unwinding dynamics of C. elegans HIM-6 using single-molecule FRET. We found that HIM-6 exhibit reiterative DNA unwinding and unwinds fewer than 31 bp without auxiliary proteins. Moreover, we determined that HIM-6 utilized the mode of “sliding-back” on the translocated strand, without strand-switching for rewinding and C. elegans RPA, single-stranded DNA binding protein, suppresses the reiterative behavior. Second, we investigated the unwinding dynamics of C. elegans WRN-1 using single-molecule FRET. WRN-1 also shows reiterative DNA unwinding and unwinds fewer than 31 bp like HIM-6. We found that WRN-1 uses “sliding-back” mode when DNA is rewound. In addition, we determined that WRN-1 uses both of DNA tails during the waiting stage before the next unwinding event. Third, we constructed the protein fragments of C. elegans HIM-6 and WRN-1 to identify a domain which is responsible for fragments. Core helicase, core-RQC, core-RQC-HRDC domains of HIM-6 and WRN-1 were cloned separately. We expect to identify the domain through smFRET measurements.