Mycobacterium tuberculosis UvrD1 and UvrA Proteins Suppress DNA Strand Exchange Promoted by Cognate and Noncognate RecA Proteins

Abstract

DNA helicases are present in all kingdoms of life and play crucial roles in processes of DNA metabolism such as replication, repair, recombination, and transcription. To date, however, the role of DNA helicases during homologous recombination in mycobacteria remains unknown. In this study, we show that Mycobacterium tuberculosis UvrD1 more efficiently inhibited the strand exchange promoted by its cognate RecA, compared to noncognate Mycobacterium smegmatis or Escherichia coli RecA proteins. The M. tuberculosis UvrD1(Q276R) mutant lacking the helicase and ATPase activities was able to block strand exchange promoted by mycobacterial RecA proteins but not of E. coli RecA. We observed that M. tuberculosis UvrA by itself has no discernible effect on strand exchange promoted by E. coli RecA but impedes the reaction catalyzed by the mycobacterial RecA proteins. Our data also show that M. tuberculosis UvrA and UvrD1 can act together to inhibit strand exchange promoted by mycobacterial RecA proteins. Taken together, these findings raise the possibility that UvrD1 and UvrA might act together in vivo to counter the deleterious effects of RecA nucleoprotein filaments and/or facilitate the dissolution of recombination intermediates. Finally, we provide direct experimental evidence for a physical interaction between M. tuberculosis UvrD1 and RecA on one hand and RecA and UvrA on the other hand. These observations are consistent with a molecular mechanism, whereby M. tuberculosis UvrA and UvrD1, acting together, block DNA strand exchange promoted by cognate and noncognate RecA proteins.

Important Role for Mycobacterium tuberculosis UvrD1 in Pathogenesis and Persistence apart from Its Function in Nucleotide Excision Repair

“…UvrD homologues can also act as translocases, and in this way they are able to displace proteins bound to DNA (1). It is possible that translocase activity of M. tuberculosis UvrD1 could contribute to clearing RecA from the DNA at fork structures (9,46). This behavior would be similar to that of the E. coli RecG helicase, which has been shown to be involved in the recovery of stalled replication forks (41).…”

“…It is thought that this action is at least partly mediated by the ability of these enzymes to restrict the actions of RecA at blocked replication forks (1,31). Indeed, biochemical studies have shown that M. tuberculosis UvrD1 suppresses DNA strand exchange reactions catalyzed by RecA in vitro (46). UvrD homologues can also act as translocases, and in this way they are able to displace proteins bound to DNA (1).…”

UvrD2 Is Essential in Mycobacterium tuberculosis, but Its Helicase Activity Is Not Required

“…The apparent requirement for ATP hydrolysis and/or DNA translocation, but not helicase, activity suggests that the essential function of UvrD2 is most likely to involve some such DNA translocation and, potentially, protein displacement. Mycobacterial UvrD1 can function to inhibit RecA-mediated strand exchange (28); whether UvrD2 also plays a role in regulating recombination remains to be seen. The identification of the crucial role of UvrD2 in M. tuberculosis growth and survival will require additional studies.…”

“…Thus, it would appear that it is UvrD1 that functions in NER in M. tuberculosis. Interestingly, M. tuberculosis UvrD1 also suppresses DNA strand-exchange reactions catalyzed by RecA (28), and M. smegmatis UvrD1 appears to play a role in regulating recombination (10), suggesting that mycobacterial UvrD1 has further roles outside NER.…”

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

“…Co-immunoprecipitation-Assays were performed as described earlier (46). Whole-cell lysates (in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.5 mM EDTA) from E. coli cells expressing MtRecD and MtRecA were treated with 50 g/ml DNase I at 37°C for 1 h. The interaction between proteins in the wholecell lysates was facilitated by incubation with gentle stirring for 6 h at 4°C.…”