As a stable double-strand molecule harboring genetic information, DNA is always damaged by endogenously physiological conditions in cells and exogenously environmental influences 1, 2, 3. Generally, DNA damages occur with modifying base or sugar and breaking single- or double-strand as well [2]. Since they produce mutation(s) or disrupt genetic information transmission, which causes genome instability, DNA damages must be promptly and accurately repaired to protect genetic information stored in genomic DNA once they have been created. Currently, cells have evolved several repair pathways for maintaining genome stability and cellular function: mismatch repair, base excision repair (BER), nucleotide excision repair and homologous recombination repair [4]. Notably, mutations in these DNA repair pathways will lead to cellular senescence or even cellular death [5].

Base deamination is a typical pathway for causing DNA damages by the hydrolytic reactions of normal bases, among which cytosine, adenine, and guanine are deaminated to form uracil, hypoxanthine, and xanthine, respectively. These base deaminations can occur spontaneously and frequently under physiological conditions, and also are accelerated by aerobic respiration, nitrosative stress, high temperature and ionizing radiation 6, 7, 8, 9, 10, 11. Before repair, replication of deaminated base will always result in mutation. For example, the GC→A:T and A:T→G:C mutations would arise after replication of uracil and hypoxanthine in DNA since uracil and hypoxanthine preferentially pair with adenine and cytosine, respectively. Notably, these mutations are present frequently in the genomes of cancer patients [12]. Thus, deaminated bases must be repaired to avoid mutations created by their replication before repair [13].

Fortunately, organisms possess a common BER for restoring deaminated base to normal base. The BER pathway is triggered with a DNA glycosylase and then is completed by other enzymes or proteins [14]. In addition to BER, alternative excision repair (AER), which is initiated with cleaving a phosphodiester bond around a lesion site by an endonuclease [15], has been proposed for DNA damage repair [16]. Endonuclease V (EndoV) can cleave the second phosphodiester bond on the 3′-side of the hypoxanthine lesion, which is the first reported endonuclease that is responsible for AER [17]. The EndoV homologs are widespread in bacteria, eukarya and Archaea [18]. Besides, another endonuclease was identified from the hyperthermophilic euryarchaeon Pyrococcus fusious, capable of cleaving DNA containing a deaminated base or an AP (apurinic/apyrimidinic) site, and this endonuclease was designated as endonuclease Q (EndoQ) [19]. In contrast with EndoV, EndoQ is only present in some Archaea, especially in Thermococcales, and in a small group of bacteria [19]. Besides, EndoQ cleaves the DNA strand at the 5′- side of the lesion [19], which also sharply contrasts with EndoV.

As the third domain of life, Archaea represent a simplified version of eukarya with respect to DNA repair since they possess the eukaryote-like DNA repair proteins 20, 21. A number of structural and biochemical data of archaeal DNA repair proteins have provided important insights into eukaryotic DNA repair [23]. Hyperthermophilic Archaea (HA) are an important branch of Archaea, which thrive in high-temperature environments. The genomic stability of HA is severely threatened since their living high-temperature environments lead to the increased base deamination and depurine or depyrimide [24]. Surprisingly, HA possess spontaneous mutation rates that are similar or lower than other organisms despite their living in inhospitable environments 25, 26. To maintain genomic DNA stability, HA must possess more highly efficient DNA repair than other organisms.

To date, HA have evolved several pathways for deaminated DNA repair. Firstly, HA possess the BER enzymes or proteins present in other organisms [21], which indicates that BER is an important pathway for deaminated DNA repair. Additionally, the genomes of all the sequenced HA encode an EndoV homolog that can act on hypoxanthine-containing DNA, thereby suggesting another potential pathway for hypoxanthine repair. Currently, the physiological role of archaeal EndoV has not been confirmed in vivo. Besides, an endonuclease NucS from HA was identified as a repair enzyme that can cleave DNA harboring a deaminated base in a manner of a Restriction Endonuclease (RE), thus providing an alternative pathway for repairing deaminated bases [27]. Furthermore, some HA encode the EndoQ homolog that cleaves DNA containing a deaminated base, which indicates another potential pathway for deaminated DNA repair [19].

Since EndoQ was originally identified from P. furiosus (Pfu-EndoQ) [19], the EndoQ orthologs have been reported from the hyperthermophilic euryarchaeon Thermococcus kodakarensis, the methanogen Methanosarcina acetivorans and the bacterium Bacillus pumilus 19, 28, 29. The common characteristic of these EndoQ homologs is that they can cleave DNA containing a deaminated base or an AP site. In addition, the crystal structures of apo Pfu-EndoQ and its complex with dsDNA containing a deaminated base or an AP site were solved 30, 31, thus providing important insight into structural and functional relationship of EndoQ. Furthermore, biochemical characteristics of Pfu-EndoQ were dissected [32].

Due to the limited reports, our understanding on biochemical characteristics and catalytic mechanism of archaeal EndoQ remains incomplete. Thermococcus gammatolerans, which was isolated from the hydrothermal vent located in the Gulf of California, is a hyperthermophilic euryarchaeon that grows optimally at 88°C [33]. This archaeon can withstand 5.0 kGy dose of gamma irradiation, which has been thought as the most radioresistant archaeon to date [34]. The genome of T. gammatolerans encodes a putative EndoQ (Tga-EndoQ). In this work, we describe biochemical characteristics of cleaving deaminated DNA by Tga-EndoQ, demonstrating that this endonuclease can cleave DNA containing a uracil, a hypoxanthine or an AP site. This EndoQ displays biochemical characteristics distinct from Pfu-EndoQ although they possess 70% amino acid similarity. Notably, the roles of six conserved residues in Tga-EndoQ were dissected by mutational analyses, demonstrating taht residues E167 and H195 in Tga-EndoQ are essential for catalysis, residues S18 and R204 in Tga-EndoQ are involved in catalysis, and residues N191 and Y299 might play structural roles.