NurA Is Endowed with Endo- and Exonuclease Activities that Are Modulated by HerA: New Insight into Their Role in DNA-End Processing

Abstract

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. However, contradictory results have been reported on the exact activities and mutual dependence of these two enzymes. To understand the functional relationship between these two enzymes we deeply characterized Sulfolobus solfataricus NurA and HerA proteins. We found that NurA is endowed with exo- and endonuclease activities on various DNA substrates, including linear (single-stranded and double stranded) as well as circular molecules (single stranded and supercoiled double-stranded). All these activities 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 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.

Resistance to UV Irradiation Caused by Inactivation ofnurAandherAGenes in Thermus thermophilus

“…T. thermophilus NurA also had nicking activity to circular dsDNA in an HerAindependent manner (Fig. 12E and G), and this characteristic is in agreement with that of D. radiodurans and S. solfataricus NurA (8,17). Similarly, T. thermophilus HerA retained ATPase activity, which was required for restoring UV sensitivity of nurA and herA disruptants to WT levels.…”

“…The molecular functions of archaeal NurA and HerA have been analyzed in detail. NurA has Mn 2ϩ -dependent 5=-to-3= exonuclease/endonuclease activities to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) (6)(7)(8). HerA is a bipolar (5=¡3= and 3=¡5=) helicase driven by ATP hydrolysis activity, which is promoted by binding to ssDNA and dsDNA (9).…”

“…NurA forms the toroidal dimer (PDB codes 3TAZ and 2YGK) (10,11) and interacts with the hexameric ring of HerA (PDB code 4D2I), which is mediated by the N-terminal HAS (HerA and ATP synthase) domain (11)(12)(13). The NurA-HerA interaction contributes to high 5=¡3= exonuclease activity of NurA (8,10,11,14). In addition, HerA proteins of Sulfolobus acidocaldarius and Sulfolobus tokodaii interact with Mre11 and/or Rad50 (15,16).…”

The Finely Coordinated Action of SSB and NurA/HerA Complex Strictly Regulates the DNA End Resection Process in Saccharolobus solfataricus

“…Furthermore, in Figure 7B, it is possible to observe a weak nuclease activity, pointed out by the arrow, due to the presence of NurA. As already demonstrated by our group [28], the nuclease activity of NurA requires Mn 2+ ions even if Mg 2+ ions, which are present in the helicase buffer (see Section 3), enable a faint nuclease activity.…”

Archaeal DNA Repair Mechanisms

“…In Sulfolobus, HerA resectioning is required for cell viability with the functional HerA complex existing as a mixture of hexameric and heptameric states bound around strands of dsDNA. The nuclease NurA is thought to preferentially bind on the outside of the hexameric HerA-dsDNA substrate, where ATP-dependent helicase activity of the HerA ring is thought to stimulate NurA activity, likely by coupling translocation and ssDNA substrate presentation for NurA to degrade [44][45][46][47]. How this complex is specifically activated by Mre11/Rad50 after recognition of DSBs to produce appropriate resectioning remains elusive and is vitally important information for understanding the initiation of DSB repair by HR.…”