An alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease

Abstract

Recent studies show that NucS endonucleases participate in mismatch repair in several archaea and bacteria. However, the function of archaeal NucS endonucleases has not been completely clarified. Here, we describe a NucS endonuclease from the hyperthermophilic and radioresistant archaeon Thermococcus gammatolerans (Tga NucS) that can cleave uracil (U)-and hypoxanthine (I)-containing dsDNA at 80 o C. Biochemical evidence shows that the cleavage sites of the enzyme are at the second phosphodiester on the 5′-site of U or I, and at the third phosphodiester on the 5′-site of the opposite base of U or I, creating a double strand break with a 4-nt 5′overhang.The ends of the cleaved product of Tga NucS are ligatable, possessing 5′phosphate and 3′-hydroxyl termini, which can be utilized by DNA repair proteins or enzymes. Tga NucS displays a preference for U/G-and I/T-containing dsDNA over other pairs with U or I, suggesting that the enzyme is responsible for repair of U and I in DNA that arise from deamination. Biochemical characterization of cleaving U-and I-containing DNA by Tga NucS was also investigated. The DNA-binding results show that the enzyme exhibits a higher affinity for normal, U-and I-containing dsDNA than for normal, U-and I-containing ssDNA. Therefore, we present an alternative pathway for repair of deaminated bases in DNA triggered by archaeal NucS endonuclease in hyperthermophilic archaea.

New Insights Into DNA Repair Revealed by NucS Endonucleases From Hyperthermophilic Archaea

“…Recently, our lab characterized the NucS endonuclease from the radioresistant hyperthermophilic euryarchaeon Thermococcus gammatolerans (Tga NucS). In addition to previously described activities of G and T mismatched DNA repair, we found that the Tga NucS can also recognize and cleave DNA with deaminated bases (uracil and hypoxanthine; Figure 1C; Zhang et al, 2020). Despite a very close proximity with Tko EndoMS (86% similarity), Tga NucS displays very divergent capabilities.…”

“…Firstly, Tga NucS can cleave U-and I-containing dsDNA on both strands to produce a double strand break with a 4-nt 5'-overhang ( Figure 1C), rather than a 5-nt 5'-overhang as observed for Tko EndoMS (Ishino et al, 2016). Both Tko EndoMS and Tga NucS can cleave hypoxanthine-containing DNA with the same efficiency as G/T mismatch (Ishino et al, 2016;Zhang et al, 2020). Tga NucS is more thermoactive than Tka EndoMS with a higher activity at physiological temperature, e.g., 80°C (Zhang et al, 2020), when Tko EndoMS works optimally at 55°C (Ishino et al, 2016).…”

“…Pyrococcus abyssi Branched and splayed DNA (Ren et al, 2009) Thermococcus kodakarensis Mismatched DNA; I-containing dsDNA (Ishino et al, 2016) Thermococcus gammatolerans U-and I-containing dsDNA; branched and splayed DNA; mismatched DNA (Zhang et al, 2020) potential role in the recognition of helix-distorting DNA damage in their putative NER pathway, including the NucS endonuclease. NucS endonuclease from the hyperthermophilic euryarchaeon P. abyssi was first characterized in vitro, demonstrating that it can cleave the branched-structure DNA substrates that contain flapped and splayed DNA by removing the large ssDNA segments at the branch points ( Figure 1A; Ren et al, 2009;Creze et al, 2011Creze et al, , 2012.…”

“…Both Tko EndoMS and Tga NucS can cleave hypoxanthine-containing DNA with the same efficiency as G/T mismatch (Ishino et al, 2016;Zhang et al, 2020). Tga NucS is more thermoactive than Tka EndoMS with a higher activity at physiological temperature, e.g., 80°C (Zhang et al, 2020), when Tko EndoMS works optimally at 55°C (Ishino et al, 2016). Taken together, it is clear that the Tga NucS exhibits several distinct biochemical properties from canonical NucS/EndoMS, which lead us to propose that Tga NucS might be involved in a novel alternative pathway for the repair of deaminated bases (uracil and hypoxanthine), in addition to BER and AER.…”

Characterization of a Family IV uracil DNA glycosylase from the hyperthermophilic euryarchaeon Thermococcus barophilus Ch5

“…Recently, an endonuclease was identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus, capable of cleaving the 5'-end DNA phosphodiester bond of uracil (8). Furthermore, a NucS endonuclease from hyperthermophilic euryarchaeon Thermococcus gammatolerans can also cleave uracil-containing DNA, potentially providing another alternative pathway for repair uracil in DNA (9). Therefore, hyperthermophilic archaea possess various pathways to repair uracil in DNA.…”