Identification and characterization of a human DNA glycosylase for repair of modified bases in oxidatively damaged DNA

Abstract

8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase/AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase/AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replication-associated repair of oxidized bases.

Figure 1

Sequence alignment of hNEH1 and hNEH2 with E. coli MutM/Nei. Identical residues are underlined. Box A contains catalytic N-terminal Pro; box B, helix-2-turn-helix motif; box C, Zn-finger motif. Potential nuclear localization signal (NLS; by psort) in NEH1 is underlined. N-terminal Met is cleaved after synthesis.

Figure 2

Trapping analysis of recombinant NEH1 in extracts of mutM nei E. coli. Lane 1, no protein; lanes 2–4, trapping assay with lysates (1 μg) of E. coli containing empty vector, WT NEH1, and Pro 1 deletion mutant, after incubation with 0.1 pmol of DHU⋅A-containing duplex oligo; lane 5, trapped complex of purified Nth (5 ng) used as a reference with 0.1 pmol of DHU⋅A. The positions of trapped complexes and free DNA are indicated.

Figure 3

Expression and purification of NEH1. Lysates (15 μg) of E. coli expressing NEH1 were analyzed by SDS/PAGE and Coomassie blue staining. The Bio-Rad protein markers are shown on the left. Purified WT NEH1 (lane 1); lanes 2–4, lysates of E. coli with empty vector, WT, and Pro-1 mutant NEH1 expression plasmid, respectively. The position of NEH1 is indicated by arrow.

Figure 4

Excision of FapyA (□) and FapyG (○) by hNEH1 from irradiated DNA. Other details are described in Materials and Methods.

Figure 5

Substrate specificity and AP lyase activity of purified hNEH1. (A) Purified NEH1 (0.5 pmol) was incubated with 5′ ³²P-labeled DHU-containing duplex oligo (4 pmol) with A, T, G, and C in the complementary strand (lanes 4–7) opposite DHU. Lanes 2 and 3, Incision activity of Nth and Nei, respectively, with DHU⋅A oligo. (B) Incision activity of NEH1 with 8-oxoG⋅A-, 8-oxoG⋅T-, 8-oxoG⋅G-, and 8-oxoG⋅C-containing oligos, respectively (lanes 2–5). Incision activity of 8-oxoG⋅C with hOGG1 and MutM (lanes 6 and 7). Positions of β and βδ-elimination products are indicated. (C) Cleavage of 5-OHU-containing oligo (100 fmol) by NEH1. Lane 1, No enzyme; lane 2, 1 pmol NEH1.

Figure 6

Tissue-specific expression of NEH1. The blots containing 2 μg poly(A)⁺ RNA purified from various human tissues (CLONTECH) were probed sequentially for Northern analysis with ³²P-labeled cDNAs of hNEH1 (Upper) and hOGG1 (Lower).

Figure 7

S phase-specific activation of NEH1. (A) Northern analysis of NEH1 mRNA, and (B) Western analysis of NEH1 polypeptide in synchronized MRC5 cells collected at various times after serum addition. The percentage of S phase cells in these different experiments is shown.