De Falco Mariarosaria, Catalano Federico, Rossi Mosè, Ciaramella Maria, De Felice Mariarita
Institute of Biosciences and Bioresources, Consiglio Nazionale delle Ricerche, Naples, 80131, Italy.
PLoS One. 2015 Nov 11;10(11):e0142345. doi: 10.1371/journal.pone.0142345. eCollection 2015.
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.
核酸酶NurA和ATP酶HerA存在于所有已知的嗜热古菌中，并与高度保守的MRE11/RAD50蛋白协同作用，以促进同源重组修复过程中高效的DNA双链断裂末端加工。然而，关于这两种酶的确切活性和相互依赖性，已有相互矛盾的报道。为了了解这两种酶之间的功能关系，我们深入研究了嗜热栖热菌的NurA和HerA蛋白。我们发现，NurA对各种DNA底物具有核酸外切酶和核酸内切酶活性，包括线性（单链和双链）以及环状分子（单链和超螺旋双链）。所有这些活性并不严格依赖于HerA的存在，需要二价离子（最好是Mn2+），并且会受到ATP存在的抑制。核酸内切酶和核酸外切酶活性有不同的要求：虽然HerA刺激线性DNA片段上的核酸外切酶活性，且该活性依赖于催化性的D58残基，但环状双链DNA上的核酸内切酶活性不依赖于HerA，且不受D58A突变的影响。基于我们的研究结果，我们提出了NurA/HerA复合物在DNA末端加工过程中的作用机制。
