Role of Mycobacterium tuberculosis DinG and UvrD helicases in unwinding G-quadruplex DNA

Abstract

Recent studies have demonstrated the role of E. coli UvrD as a replicative helicase that facilitates replication of rolling circle plasmids. Along with dinG and rep helicase, UvrD a so-called “accessory helicase” removes protein roadblocks such as the transcription apparatus by pulling back RNA polymerase and recruiting the DNA repair machinery, thereby assisting genome duplication. In our earlier study, we demonstrated that M. tuberculosis DinG unwinds tetra and bi molecular G4 DNA structures. Rep, DinG and UvrD accessory helicases have overlapping functions and two out of three helicases are required for the viability of E. coli with inverted rrn operon. The RecQ and Rep helicases are absent in M. tuberculosis. Moreover, UvrD and DinG have overlapping functions with respect to replication and transcription in E. coli. Hence we wanted to investigate the role of M. tuberculosis UvrD helicase in G4 DNA resolution. M. tuberculosis contains two copies of UvrD: UvrD1 and UvrD2 which shares 40% and 33% amino acid sequence identity with E. coli UvrD respectively. We cloned, overexpressed and purified M. tuberculosis UvrD1 and UvrD2 helicases from E. coli BL21(*DE3) cells to near homogeneity. The purified MtUvrD1 exhibited 3ˈ5ˈ translocase and helicase activity. The unwinding of TP-G4 and OX1T G4 DNA by MtUvrD1 was ATP and Mg2+ dependent and requires a minimum of 7 nts at the 3ˈ single stranded region. Interestingly, we found that MtUvrD1 showed 3 fold less helicase activity with replication intermediate substrates compared to G4 DNA substrates. Many of the mycobacterial gene promoters and inter- or intragenic regions contain G4 DNA forming motifs. We found that MtUvrD1 unwinds tetramolecular G4 structures that are formed from the intragenic G4 structures of recA, trpD and MSF transporter genes. Several G4 specific ligands have been shown to effectively bind to G4 DNA and inhibit resolution by helicases. Our data shows TMPyP4 and NMM could specifically bind recA and MSF G4-DNA and inhibit unwinding by MtUvrD1. MtUvrD2 showed increase in G4-DNA unwinding with increase in 3ˈ tail length.