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Abstract
Generation of the 3′ overhang is a critical step during homologous recombination (HR) and replication fork rescue processes. This event is usually performed by a series of DNA nucleases and/or helicases. The nuclease NurA and the ATPase HerA, together with the highly conserved MRE11/RAD50 proteins, play an important role in generating 3′ single-stranded DNA during archaeal HR. Little is known, however, about HerA-NurA function and activation of this fundamental and complicated DNA repair process. Herein, we analyze the functional relationship among NurA, HerA and the single-strand binding protein SSB from Saccharolubus solfataricus. We demonstrate that SSB clearly inhibits NurA endonuclease activity and its exonuclease activities also when in combination with HerA. Moreover, we show that SSB binding to DNA is greatly stimulated by the presence of either NurA or NurA/HerA. In addition, if on the one hand NurA binding is not influenced, on the other hand, HerA binding is reduced when SSB is present in the reaction. In accordance with what has been observed, we have shown that HerA helicase activity is not stimulated by SSB. These data suggest that, in archaea, the DNA end resection process is governed by the strictly combined action of NurA, HerA and SSB.
Highlights
In all organisms, the accurate and faithful duplication of the genomic DNA depends on the joint work of DNA repair and genetic recombination machineries
The long 3 -ssDNA tails were generated coated by multiple copies of the replication protein A (RPA) that form a filament on the ssDNA to prevent the formation of secondary structures
homologous recombination (HR) is one of the most important double-strand breaks (DSBs) repair pathways and, in contrast to non-homologous end joining (NHEJ), it is a high-fidelity mechanism since it relies upon homologous DNA sequences and generates error-free repaired products
Summary
The accurate and faithful duplication of the genomic DNA depends on the joint work of DNA repair and genetic recombination machineries. While NHEJ can occur throughout the cell cycle, HR is limited to S and G2 phases because of the presence of the homologous copy of the damaged DNA The choice between these two pathways is dictated by a DNA mechanism known as DNA end resection, a tightly regulated machinery that ensures genomic stability [4,5]. During this process, DNA ends are resected through the joint action of helicases and nucleases that unwind the DNA duplex and generate 3 overhangs required in S and G2 phases for the subsequent repair steps of HR [6,7]. In order to gain further insight into S. solfataricus NurA and NurA/HerA complex roles in the DNA end resection process, we performed an in vitro characterization of their DNA nuclease, helicase and DNA binding activities in combination with S. solfataricus SSB protein. We propose a hypothetical model of DNA end resection mechanism in S. solfataricus, which could lead to a better understanding of this process even in higher organisms, such as eukaryotes
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