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.
Highlights
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
Initiation of homologous recombination requires the processing of DNA ends in 3’ overhangs, which are required for recombinase, loading and subsequent strand invasion
Our study is focused on the analysis of the mechanism of DNA end-resection in S. solfataricus and in particular we investigated on NurA nuclease activity alone and in complex with HerA
Summary
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. NurA and DNA-End Processing 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 process in which the two ends of the broken chromosome are ligated back together directly. HR is one of the most important DSB repair pathways [1,2] and, in contrast to NHEJ, it is a high-fidelity mechanism since it relies upon homologous DNA sequences and generates error-free repaired products. During HR initiation, enzymatic resection of DNA ends generates 30-single-stranded DNA (ssDNA) overhangs that are necessary for loading recombinases (RecA/Rad51/RadA) [3,4]
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