Structural and functional properties of CiNTH, an endonuclease III homologue of the ascidian <i>Ciona intestinalis</i>: critical role of N-terminal region

Abstract

Oxidatively damaged bases in DNA can cause cell death, mutation and/or cancer induction. To overcome such deleterious effects of DNA base oxidation, cells are equipped with base excision repair (BER) initiated by DNA glycosylases. Endonuclease III (Nth), a major DNA glycosylase, mainly excises oxidatively damaged pyrimidines from DNA. The aims of this study were to obtain an overview of the repair mechanism of oxidatively damaged bases and to elucidate the function of BER in maintaining genome stability during embryogenesis and development. In this study, we used the ascidian Ciona intestinalis because at every developmental stage it is possible to observe the phenotype of individuals with DNA damage or mutations. Sequence alignment analysis revealed that the amino acid sequence of Ciona intestinalis Nth homologue (CiNTH) had high homology with those of Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and human Nth homologues. It was evident that two domains, the Helix-hairpin-Helix and 4Fe-4S cluster domains that are critical regions for the Nth activity, are well conserved in CiNTH. CiNTH efficiently complemented the sensitivity of E. coli nth nei mutant to H 2 O 2 . CiNTH was bifunctional, with DNA glycosylase and AP lyase activities. It removed thymine glycol, 5-formyluracil and 8-oxoguanine paired with G from DNA via a β-elimination reaction. Interestingly, the N-terminal 44 amino acids were essential for the DNA glycosylase activity of CiNTH.

Low-intensity red and infrared laser effects at high fluences on Escherichia coli cultures

“…However, high fluences of red and infrared lasers decrease the survival rates of exponential phase E. coli cultures deficient in endonuclease III (JW1625-1) and E. coli cultures deficient in endonuclease IV (JW2146-1) ( Table 2 ). As part of its base excision repair mechanism, endonuclease III repairs apurinic/apyrimidinic sites and damaged pyrimidines ( 19 , 20 ). Similarly, endonuclease IV acts on apurinic/apyrimidinic sites and oxidatively damaged bases in E. coli ( 21 22 23 24 ).…”

A novel DNA damage response mediated by DNA mismatch repair in Caenorhabditis elegans: induction of programmed autophagic cell death in non-dividing cells

“…Mitomycin C (MMC) is a cross-linking agent that generates interstrand cross linking in genomic DNA [ 35 ]. Methyl viologen (MV), which is also called paraquat, is a superoxide-inducing agent, and generates oxidative DNA lesions in genomic DNA [ 36 , 37 ]. Sodium hydrogen sulfite (NaHSO 3 ) is a deaminating agent that generates deaminated DNA lesions such as uracil and 5-(hydroxymethyl) uracil in genomic DNA [ 38 ].…”

CiApex1 has AP endonuclease activity and abrogated AP site repair disrupts early embryonic development in <i>Ciona intestinalis</i>

“…Expression and purification of CiApex1 and DN-CiApex1 protein Expression and protein purification were performed according to previously described protocols with some modifications (Jin et al, 2006;Kato et al, 2012). To avoid endogenous AP endonuclease contamination, AP endonuclease-deficient E. coli RPC501 (xth − nfo − ) was used as a host strain for purifying GST-CiApex1 and GST-DN-CiApex1 protein.…”

“…Detection of AP endonuclease activity AP endonuclease activity was detected according to previously described protocols with some modifications (Jin et al, 2006;Kato et al, 2012). The sequences of the oligonucleotides used in this study are shown in Fig.…”

“…A previous study suggested that CiApex1, a C. intestinalis homolog of human APEX1 (hAPEX1), was expressed during early development of C. intestinalis (El-Mouatassim et al, 2007). Furthermore, homologs of the DNA glycosylases Ogg1 and Nth have already been identified and characterized in C. intestinalis (Jin et al, 2006;Kato et al, 2012), and were suggested to participate in base excision repair.…”