A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus

Abstract

DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:T→G:C transition mutation during DNA replication. Endonuclease V (EndoV) hydrolyzes the second phosphodiester bond 3′ from deoxyinosine in the DNA strand, and was considered to be responsible for hypoxanthine excision repair. However, the downstream pathway after EndoV cleavage remained unclear. The activity to cleave the phosphodiester bond 5′ from deoxyinosine was detected in a Pyrococcus furiosus cell extract. The protein encoded by PF1551, obtained from the mass spectrometry analysis of the purified fraction, exhibited the corresponding cleavage activity. A putative homolog from Thermococcus kodakarensis (TK0887) showed the same activity. Further biochemical analyses revealed that the purified PF1551 and TK0887 proteins recognize uracil, xanthine and the AP site, in addition to hypoxanthine. We named this endonuclease Endonuclease Q (EndoQ), as it may be involved in damaged base repair in the Thermococcals of Archaea.

A functional endonuclease Q exists in the bacterial domain: identification and characterization of endonuclease Q from Bacillus pumilus

“…In addition to Thermococcales and other methanogens in the archaeal domain, the EndoQ homologs were identified from bacterial domain: Actinobacteria, Firmicutes, Proteobacteria, and Spirochaetes, although homologs in Actinobacteria were not found in the previous study. 8) Among the bacterial sequence, we selected the EndoQ homolog in B. pumilus and cloned the gene from the strain 8G134 as described in Materials and Methods. The encoded protein, designated BpuEndoQ, presented 32% amino acid identity to PfuEndoQ and the critical residues for the nuclease activity are highly conserved (Fig.…”

“…DNA substrates were obtained and prepared as described before. 8) The cleavage reactions were performed at 30°C for 10 min in a 20 μL reaction mixture, containing 50 mM Tris-HCl, pH8.0, 1 mM DTT, 1 mM MgCl 2 , 0.01% Tween20, 5 nM DNA substrate, and various concentrations of BpuEndoQ (0, 1, 10, 100 nM). Reactions were terminated with 20 μL of stop solution (98% deionized formamide, 10 mM EDTA, and 0.1% Orange G).…”

“…EndoQ from Thermococcus kodakarensis, an organism belonging to the same order as P. furiosus, showed the same biochemical properties as PfuEndoQ. 8) Further analyses showed that EndoQ and EndoV do not directly interact with each other in either the presence or absence of DNA, but they individually work on deoxyinosine (dI)-containing DNA at each cleavage site. The EndoQ pathway is probably another pathway of alternative excision repair (AER), which is initiated by a single nick near the site of a DNA lesion, in some of the organisms in Archaea.…”

“…11) The homologs of this enzyme are conserved only in Thermococcales and some of the methanogens in Archaea, and are absent from most of the members of the domains Bacteria and Eukarya. 8) The phylogenetic analysis suggested that EndoQ originated in the domain Archaea. 8) However, proteins with the homologous sequence of EndoQ are found in a small group of Bacteria, and it is of significance to determine whether the bacterial EndoQ-like polypeptides actually function as damaged base-specific endonuclease.…”

“…8) The phylogenetic analysis suggested that EndoQ originated in the domain Archaea. 8) However, proteins with the homologous sequence of EndoQ are found in a small group of Bacteria, and it is of significance to determine whether the bacterial EndoQ-like polypeptides actually function as damaged base-specific endonuclease. Here, we cloned the gene encoding a putative EndoQ in Bacillus pumilus 8G134 and reported the biochemical characterization of the purified gene product in vitro.…”