Structural And Functional Studies Of The DNA End Resection In Deinococcus Radiodurans

Deinococcus radiodurans is one of the most radioresistant bacteria in the world, whose robust DNA damage response and repair system have been studied for years. Homologous recombination (HR), the major repair pathway for double-strand breaks in bacteria, consists of RecBCD system and RecFOR system. Lacking RecBCD system naturally, D. radiodurans takes RecFOR system as the major recombinational DNA repair pathway. Therefore, D. radiodurans is regarded as the most suitable species for studying the RecFOR system. The resection of DNA strand at double-strand breaks is an essential step in recombinational DNA repair, from which a 3’-end ssDNA was created, followed by strand invasion, exchange and recombination. Not as the DNA end resection process in RecBCD system, which has been highly clarified, that process in RecFOR system has not been well characterized.The nuclease RecJ, helicase RecQ, and the single-stranded binding protein (SSB), are suggested to join in this process cooperately. In order to characterize the DNA end resection process in RecFOR system, the present work solved the crystal structures of D. radiodurans RecJ (DrRecJ) in complex with deoxythymidine monophosphate (dTMP), DNA and the C-terminal region of single-stranded DNA-binding protein (SSB-Ct), and did some related assays. The active site of RecJ in the N-terminal domain contains two divalent cations that coordinate the nucleophilic water and the ssDNA makes a 180° turn at the scissile phosphate. A terminal 5’-phosphate-binding pocket above the active site determines the 5’-3’polarity of the deoxy-exonuclease of RecJ. A helical gateway at the entrance to the active site admits ssDNA only. And the continuous stacking interactions between protein and nine nucleotides ensure the processive end resection. The C-terminal domain of RecJ binds the SSB-Ct, which explains how RecJ and SSB work together to efficiently process broken DNA-ends for homologous recombination. Together with the cooperation of RecQ helicase, we throw light upon the DNA end resection model in RecFOR system.On the other hand, we identified that the C-terminal domain of RecJ could also interact with ATPase/translocase HerA. Cooperating with nuclease NurA, HerA and NurA were suggested to participate in the DNA end resection in archaeal HR repair. Here, we clarified the biochemical and functional features of D. radiodurans HerA and NurA. drHerA is a ATPase and drNurA is a manganese-dependent 5’-3’ ssDNA/double-stranded DNA (dsDNA) exonuclease/endonuclease. These two proteins stimulated each other’s activity through direct protein-protein interactions. The N-terminal HAS domain of DrHerA was the key domain for this interaction. Several critical residues of DrNurA and DrHerA were verified by site-directed mutational analysis. Temperature-dependent activity assays confirmed that the two proteins have mesophilic features, with optimum activity temperatures 10℃ to 15℃ higher than D. radiodurans optimum growth temperatures. Knocking out either nurA or her A affected cell proliferation by shortening the growth phase, especially for growth at a high temperature (37℃), increased the Mitomycin C (MMC) resistance, to some extent. Theses phenotypes are quite opposite from the recJ mutant, which show damaged cell growth and MMC resistance. Furthermore, results of nuclease assays showed that drHerA could stimulate drRecJ activity, while the addition of drNurA blocked this stimulation. We therefore suggest that HerA-NurA, widely existing in archaea, might play some regulation roles in RecFOR system in D. radiodurans, the mechanism of which needs further study.