Crystal structure of a repair enzyme of oxidatively damaged DNA, MutM (Fpg), from an extreme thermophile,Thermus thermophilusHB8

Abstract

The MutM [formamidopyrimidine DNA glycosylase (Fpg)] protein is a trifunctional DNA base excision repair enzyme that removes a wide range of oxidatively damaged bases (N-glycosylase activity) and cleaves both the 3¢-and 5¢-phosphodiester bonds of the resulting apurinic/apyrimidinic site (AP lyase activity). The crystal structure of MutM from an extreme thermophile, Thermus thermophilus HB8, was determined at 1.9 A Ê resolution with multiwavelength anomalous diffraction phasing using the intrinsic Zn 2+ ion of the zinc ®nger. MutM is composed of two distinct and novel domains connected by a¯exible hinge. There is a large, electrostatically positive cleft lined by highly conserved residues between the domains. On the basis of the three-dimensional structure and taking account of previous biochemical experiments, we propose a DNA-binding mode and reaction mechanism for MutM. The locations of the putative catalytic residues and the two DNA-binding motifs (the zinc ®nger and the helix±two-turns±helix motifs) suggest that the oxidized base is¯ipped out from double-stranded DNA in the binding mode and excised by a catalytic mechanism similar to that of bifunctional base excision repair enzymes. Keywords: base excision repair/crystal structure/extreme thermophile/MutM protein/Thermus thermophilus HB8 IntroductionIn aerobic organisms, cellular DNA is frequently damaged by activated oxygen species from aerobic energy metabolism or oxidative stress. Highly reactive oxygen accelerates the rate of spontaneous mutation and has, therefore, been implicated as a causative agent for ageing and in the pathogenesis of disease, including cancer (Breimer, 1990;Ames et al., 1995). In particular, the 8-oxoguanine (GO) lesion is one of the most stable products of oxidative DNA damage (Dizdaroglu, 1985). GO can form a pair with adenine as well as with cytosine, resulting in a G:C to T:A transversion (Wood et al., 1990;Shibutani et al., 1991). In Escherichia coli, the GO repair system, composed of the MutM protein [EC 3.2.2.23], also called formamidopyrimidine DNA glycosylase (Fpg), accompanied by MutY and MutT, prevents this mutation (Michaels et al., 1992).The mutM (fpg) gene encoding the MutM protein is highly conserved across a wide range of aerobic bacteria. These enzymes (M r 30 kDa) possess the invariant N-terminal sequence Pro-Glu-Leu-Pro-Glu-Val-, two strictly conserved lysine residues (Lys52 and Lys147) and a zinc ®nger motif (-Cys-X 2 -Cys-X 16 -Cys-X 2 -Cys-) at the C-terminus ( Figure 1C).MutM was originally isolated from E.coli and characterized as a DNA glycosylase that removes 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Fapy), the imidazole ring-open form of N 7 -methylguanine (Boiteux et al., 1987). More recently, MutM was reported to bind to a wide range of oxidatively damaged bases (see Figure 5C), including GO paired to cytosine (Tchou et al., 1991), formamidopyrimidine (FapyG or FapyA) and 5-hydroxycytosine (5OHC) (Hatahet et al., 1994) and to apurinic/apyrimidinic (AP) sites (Castaing et al., 1992). MutM h...

The crystal structure of human endonuclease VIII-like 1 (NEIL1) reveals a zincless finger motif required for glycosylase activity

“…Although the protein construct used for this study comprises residues 2-342 (including the C-terminal hexa-His tag), there is no identifiable density beyond residue 290. A search with the DALI server (53) confirmed the expected structural similarity with other DNA glycosylases of the Fpg͞Nei family, Thermus thermophilus Fpg (TthFpg; PDB ID code 1EE8; C␣ rms deviation ϭ 2.4 Å, 29% sequence identity, Z score ϭ 21.0) (21) and EcoNei (PDB ID code 1K3W; C␣ rms deviation ϭ 3.1 Å, 25% sequence identity, Z score ϭ 17.7) (20). The numbers reported by DALI indicate that NEIL1 is structur- Rmerge ϭ ͚ ͉I Ϫ ͗I͉͘͞ ͚ I, where ͗I͘ is the average intensity from multiple observations of symmetry-related reflections.…”

“…3). There are nonetheless significant differences: A segment comprising ␣H and the loop connecting ␣G and ␣H in NEIL1 corresponds to a region that has been shown to be disordered in the borohydride trapped covalent Nei-DNA (20) and Fpg-DNA (22, 24) complexes or complexes with DNA containing an abasic site (23,24), whereas it is ordered in uncomplexed TthFpg (21). The difference, however, is not just due to the presence or absence of DNA, but seems to also hinge on the presence of a nucleobase lesion.…”

“…Fpg and Nei also share common structural motifs, including helix-two-turns-helix (H2TH) and antiparallel ␤-hairpin zinc finger motifs (6,12). A comparison of EcoNei covalently complexed to DNA (20) with the Fpg structures (21)(22)(23)(24) revealed that their overall folds are very similar; however, their substrate preferences are markedly different: EcoFpg prefers 8-oxoguanine and oxidized purines (25,26), whereas EcoNei recognizes oxidized pyrimidines (19,(27)(28)(29).…”

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

“…However, some members of these families have common npg substrates. OGG1, MutY, Nth and their homologs have HhH structure [42][43][44]; while MutM (Fpg) and Nei have H2TH motif [44][45][46][47].…”

Antimutagenic specificities of two plant glycosylases, oxoguanine glycosylase and formamidopyrimidine glycosylase, assayed in vivo

“…Gao and Murphy [2] earlier reported that FPG-2 had a limited amount of activity in vitro on depurinated, UV-treated, and methylene-blue-treated DNA (but not on 8-oxo-G-containing oligonucleotides). FPG-2 contains the N-terminal domain and the H2TH motif in the C-terminal domain, but lacks the FPG-1 C-terminal sequence that corresponds to the bacterial zinc-finger motif [13]; this motif contributes to stabilization of the enzyme-DNA complex [14]. Other products of the Arabidopsis fpg gene splicing variants found from cloning and RT-PCR screens were not tested, but most of these are predicted to lack essential active site amino acids or have other major disruptions in their C-terminal amino acid sequences [1], so it seems unlikely that they would be active.…”