The nuclease NurA and the ATPase HerA are present in all

The nuclease NurA and the ATPase HerA are present in all known thermophilic archaea and cooperate with the highly conserved MRE11/RAD50 proteins to facilitate efficient DNA double-strand break end processing during homologous recombinational repair. are not strictly dependent on the presence of HerA require divalent ions (preferably Mn2+) and are inhibited by the presence of ATP. The endo- and exonculease activities have distinct requirements: whereas the exonuclease activity on linear DNA fragments is stimulated by HerA and depends on the catalytic D58 residue the endonuclease activity on circular double-stranded DNA is HerA-independent and MC1568 is not affected by the D58A mutation. On the basis of our results we propose a mechanism of action of NurA/HerA complex during DNA end processing. Introduction In all organisms genomic DNA is continuously subjected to a wide variety of lesions; the rapid detection of the damage and the subsequent accurate repair is crucial to maintain genomic integrity. DNA lesions are generated either by external agents such as UV light mechanical stress ionizing radiation carcinogens or intrinsic errors occurring during DNA replication recombination and aberrant chromosome segregation. Among the various types of DNA lesions double-strand breaks (DSBs) are one of the most harmful because if not correctly repaired may result in chromosome loss or deletions translocations and genomic instability causing a profound influence in proliferation of normal cells and eventually cell death. In eukaryotic cells two major DSB repair pathways are known: Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). The NHEJ pathway is an error-prone procedure where the two ends from the damaged chromosome are ligated back again together straight. HR is among the most significant DSB restoration pathways [1 2 and as opposed to NHEJ it really is a Rabbit Polyclonal to EHHADH. high-fidelity system since it depends upon homologous DNA sequences and produces error-free repaired items. During HR initiation enzymatic resection of DNA ends generates 3′-single-stranded DNA (ssDNA) overhangs that are essential for launching recombinases (RecA/Rad51/RadA) [3 4 HR continues to be investigated thoroughly in bacterias and eukarya. It’s been recommended that in eukaryotes DSBs that happen in the G1 stage from the cell routine are likely to be fixed NHEJ while those happening in the S/G2 stage are preferentially prepared HR [5 6 The HR equipment is made up of a core protein complex containing Mre11-Rad50-Nbs1 (human MRN) or Mre11-Rad50-Xrs2 (and and [26] moreover the four genes are co-induced in response to UV irradiation [27 28 data support the hypothesis that these four proteins are involved in HR DNA end resection [25 29 30 products are believed to be the functional homologous of eukaryotic Exo1/EXO1 Dna2/DNA2 and Sgs1/BLM proteins since the corresponding genes have not been found in archaea so far. This hypothesis has been supported by biochemical characterization of the encoded proteins: HerA proteins characterized from a few archaeal species all exhibit ATPase activity and for some of them ([25 29 30 35 36 However contradictory results have been reported on the properties of this complex: NurA from was reported to MC1568 display MC1568 both single-stranded endonuclease and 5′→3′ exonuclease activity on single-stranded and double-stranded DNA [32]; in contrast the very similar NurA from was found to be completely inactive in the absence of HerA [30]; moreover NurA from was reported to have a weak Mn2+ dependent 5′ to MC1568 3′ exonuclease activity but no nicking activity [25 36 as also reported for the protein [37]. Recently studies demonstrated that all four genes of the operon are essential for viability and in particular the ATPase activtiy of HerA the nuclease activity of NurA and their interaction [38]. In this study we used a biochemical approach in order to clarify the properties and functional interaction of NurA and HerA from the hyperthermophilic archaeon NurA in we detected a prominent nuclease activity. Since Blackwood JK NurA purification and activity profile. NurA and HerA were homogeneously purified from E. cultures (S1 Fig) and analyzed for their oligomeric states using a Superdex 200 10/300 gel filtration column and they turned out to be.