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

Nucleic Acids Research A novel endonuclease that may be responsible for damaged DNA base repair in Pyrococcus furiosus Miyako Shiraishi 0 Sonoko Ishino 0 Takeshi Yamagami 0 Yuriko Egashira 0 Shinichi Kiyonari 0 Yoshizumi Ishino 0 0 Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University , 6-10-1 Hakozaki, Higashi-ku, Fukuoka, Fukuoka 812-8581 , Japan DNA is constantly damaged by endogenous and environmental influences. Deaminated adenine (hypoxanthine) tends to pair with cytosine and leads to the A:TG: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. - INTRODUCTION DNA is damaged by endogenous and environmental influences. Extensive studies on excision repair systems, including nucleotide excision repair and base excision repair (BER), are being performed around the world, and our knowledge is constantly increasing (14). The excision repair initiated by a single nick near the site of a DNA lesion is now referred to as alternative excision repair (AER). This type of repair starts with an endonuclease that recognizes the damaged DNA and cleaves the phosphodiester bond near the lesion site (57). Base deamination is a typical form of DNA damage. Deaminated adenine, guanine and cytosine are called hypoxanthine, xanthine and uracil, respectively. These deaminations occur spontaneously under physiological conditions, and are promoted by ionizing radiation, high temperature, aerobic respiration and nitrosative stress. The hydrogen bonding properties of the bases are altered by the amino-keto conversion derived from deamination. For example, hypoxanthine in DNA tends to pair with cytosine, but not thymine, which is the natural binding partner of adenine. This property of hypoxanthine leads to an A:TG:C transition mutation during DNA replication (8). Therefore, the hypoxanthine sites must be repaired to prevent mutations. Two major pathways, BER and AER, are known to remove the deaminated bases. The BER pathway is based on DNA glycosylase, and several enzymes belonging to the uracil DNA glycosylase (UDG) superfamily have been identified (9). On the other hand, AER is initiated by nicking at the lesion site by a specific endonuclease (5). Endonuclease V (EndoV) is well known as the enzyme responsible for cleaving the second phosphodiester bond on the 3 -side of the deaminated base lesion. EndoV, encoded by the nif gene, was originally identified in Escherichia coli as an endonuclease that nicks DNA containing a damaged base, and was subsequently proved to be a deoxyinosine (dI) 3 -endonuclease (1012). Furthermore, analyses of nif mutant strains revealed that E. coli EndoV plays a major role in dI repair in the cells, although broader substrate specificity toward mismatched base pairs, including apurinic/apyrimidinic (AP) sites, flap DNA and pseudo-Y DNA structures, was detected in vitro (1214). EndoV homologs are conserved in all three domains of life: Bacteria, Eukarya and Archaea (15,16). The endonuclease activity for DNA containing dI has been shown for the mouse and human enzymes, as the eukaryotic EndoVs (17,18). However, it has not been determined whether the AER pathway with the EndoV homolog actually functions in eukaryotic cells. The archaeal EndoVs are diverse. Archaeoglobus fulgidus EndoV (AfuEndoV) exhibits strict specificity for dI-containing substrates in vitro (19). On the other hand, the Ferroplasma acidarmanus enzyme consists of the O6-alkylguanine-DNA alkyltransferase domain and the EndoV domain (therefore, it is called FacAGT-EndoV), and shows cleavage activities for DNA substrates containing uracil, hypoxanthine and xanthine bases in vitro (20). We characterized the EndoV homolog from the hyperthermophilic euryarchaeon, Pyrococcus furiosus (PfuEndoV) and discovered its strict substrate specificity to hypoxanthine in vitro (21). To elucidate the EndoV-mediated repair pathway in archaeal cells, the proteins related to the cleavage reaction of dI-containing DNA were screened, and we identified the protein possessing the activity to cleave the phosphodiester bond 5 from dI. This novel endonuclease, designated as Endonuclease Q (EndoQ), is conserved only in Thermococcals and some of the methanogens in Archaea, and is not present in most Bacteria and Eukarya. MATERIALS AND METHODS DNA substrates The 7-deaza-2 -deoxyxanthosine (dX)-containing oligonucleotide was obtained by custom synthesis (BEX, Tokyo, Japan). The other oligonucleotides, including the dI, deoxyuridine (dU) and tetrahydrofuran (AP)-containing oligonucleotides, were obtained from Hokkaido System Science (Sapporo, Japan) and Sigma Genosys (Tokyo, Japan). The tetrahydrofuran-containing oligonucleotide (45-AP25) was used as a model compound of the AP site. Fluorescently (Cy5 or FITC) or 32P-labeled oligonucleotides were synthesized or prepared, respectively. Double-stranded DNA was prepared by incubating the oligonucleotide and its complementary oligonucleotide in TAM buffer (40-mM Tris-acetate, pH 7.8, and 0.5-mM magnesium acetate) and annealing by an incubation in a decreasing temperature gradient. The sequences of the oligonucleotides and the oligonucleotide pairs for dsDNA are shown in Supplementary Table S1. P. furiosus cell cultivation and cell extract fractionation P. furiosus was cultivated with agitation at 98C in 4 l of medium, containing 10-g Bacto Tryptone (BD), 5-g Bacto Yeast Extract (DIFCO), 38-g Marine Art SF-1 (Osaka Yakken; consisting of 22.1-g NaCl, 9.9-g MgCl2, 1.5-g CaCl22H2O, 3.9-g Na2SO4, 0.61-g KCl, 0.19-g NaHCO3, 96-mg KBr, 78-mg Na2B4O710H2O, 13-mg SrCl2, 3-mg NaF, 1-mg LiCl, 81- g KI, 0.6- g MnCl24H2O, 2- g CoCl26H2O, 8- g AlCl36H2O, 5- g FeCl36H2O, 2- g Na2WO42H2O and 18- g (NH4)6Mo7O244H2O), and 10g Soluble Starch (Nacalai Tesque) per liter. The cells were grown to an OD600 of 0.58. After cultivation, the cells were harvested by centrifugation (3315 g, for 5 min at 25C) and sonicated for 5 min in buffer A (20-mM potassium phosphate, pH 7.4, 0.1-M NaCl, 0.5-mM DTT, 0.1-mM ethylenediaminetetraacetic acid (EDTA) and 10% glycerol) containi (...truncated)