Differential Specificity of Human and Escherichia coli Endonuclease III and VIII Homologues for Oxidative Base Lesions

Abstract

In human cells, oxidative pyrimidine lesions are restored by the base excision repair pathway initiated by homologues of Endo III (hNTH1) and Endo VIII (hNEIL1 and hNEIL2). In this study we have quantitatively analyzed and compared their activity toward nine oxidative base lesions and an apurinic/apyrimidinic (AP) site using defined oligonucleotide substrates. hNTH1 and hNEIL1 but not hNEIL2 excised the two stereoisomers of thymine glycol (5R-Tg and 5S-Tg), but their isomer specificity was markedly different: the relative activity for 5R-Tg:5S-Tg was 13:1 for hNTH1 and 1.5:1 for hNEIL1. This was also the case for their Escherichia coli homologues: the relative activity for 5R-Tg:5S-Tg was 1:2.5 for Endo III and 3.2:1 for Endo VIII. Among other tested lesions for hNTH1, an AP site was a significantly better substrate than urea, 5-hydroxyuracil (hoU), and guanine-derived formamidopyrimidine (mFapyG), whereas for hNEIL1 these base lesions and an AP site were comparable substrates. In contrast, hNEIL2 recognized an AP site exclusively, and the activity for hoU and mFapyG was marginal. hNEIL1, hNEIL2, and Endo VIII but not hNTH1 and Endo III formed cross-links to oxanine, suggesting conservation of the -fold of the active site of the Endo VIII homologues. The profiles of the excision of the Tg isomers with HeLa and E. coli cell extracts closely resembled those of hNTH1 and Endo III, confirming their major contribution to the repair of Tg isomers in cells. However, detailed analysis of the cellular activity suggests that hNEIL1 has a significant role in the repair of 5S-Tg in human cells.DNA carrying vital genetic information of cells constantly suffers from spontaneous deamination and depurination, alkylation, and oxidation (1-3). These reactions lead to modifications of the DNA backbone and bases, with the latter predominating. The resulting aberrant bases are potentially genotoxic because of the loss or alteration of base pairing information (4), and hence need to be restored by the cellular repair system (2, 3, 5). The major repair mechanism for such damage is the base excision repair (BER) 1 pathway (6), which is conserved from bacteria to humans. In the first step of BER, DNA glycosylases with distinct damage specificities detect the aberrant base in the vast sea of normal bases and remove it from the DNA backbone, leaving an apurinic/apyrimidinic (AP) site. The resulting AP site is further processed and repaired by the subsequent action of AP endonuclease (Endo), DNA polymerase, and DNA ligase through the short patch or long patch BER pathway.The initial search for DNA glycosylases involved in the repair of oxidatively damaged bases in Escherichia coli identified Endo III, Endo VIII, and formamidopyrimidine-DNA glycosylase (7,8). The principal substrates of Endo III and Endo VIII are oxidative pyrimidine lesions. They exhibit redundant damage specificity and catalyze the hydrolysis of the N-glycosidic bond (N-glycosylase activity) and the subsequent incision of an AP site by AP lyase activity via ␤-elimination ...

Effects of base excision repair proteins on mutagenesis by 8-oxo-7,8-dihydroguanine (8-hydroxyguanine) paired with cytosine and adenine

“…The findings reported herein provides the first evidence to demonstrate that NTH1 and NEIL1 suppress the mutagenesis induced by 8-oxo-Gua in living cells. It has been suggested that NTH1 binds to ds DNA containing an 8-oxo-Gua:C pair, but does not excise the 8-oxo-Gua base in vitro [39,40]. The activity of NTH1 for thymine glycol is enhanced in the presence of p53, XPG, YB-1, and APE1 [52][53][54].…”

“…NEIL1 has the ability to excise not only 8-oxo-Gua in ds DNA [39,[41][42][43] but also 8-oxo-Gua in ss DNA [44]. In addition, proliferating cell nuclear antigen (PCNA) interacts with NEIL1 and stimulates NEIL1 activity with respect to excising 5-hydroxyuracil from ss DNA sequences, including fork structures [55].…”

“…In addition, the activity of NEIL1 for the 8-oxo-Gua:C pair, as well as 8-oxo-Gua in single-stranded (ss) DNA, is very low [39,[41][42][43][44]. However, these results may not reflect the in vivo situation, where its activity could be regulated by the presence of other proteins.…”

“…It has been reported that the overexpression of OGG1 and MUTYH suppress mutations by 8-oxo-Gua in cultured cells [30,31]. The NTH1 and NEIL1 proteins are homologues of Escherichia coli Nth and Nei, DNA glycosylases that are specific for oxidized pyrimidine bases [32][33][34][35][36][37][38][39]. The observation that NTH1 can be efficiently trapped to an oligonucleotide duplex containing 8-oxo-Gua:C by treatment with NaBH 4 , suggests that NTH1 binds to 8-oxo-Gua:C DNA [40].…”

“…The observation that NTH1 can be efficiently trapped to an oligonucleotide duplex containing 8-oxo-Gua:C by treatment with NaBH 4 , suggests that NTH1 binds to 8-oxo-Gua:C DNA [40]. However, no evidence has been reported to show that NTH1 catalyzes the cleavage of 8-oxo-Gua:C oligonucleotides in vitro [39,40].…”