New family of deamination repair enzymes in uracil-DNA glycosylase superfamily

Abstract

DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigations identified five families within the uracil-DNA glycosylase (UDG) superfamily. All enzymes within the superfamily studied thus far exhibit uracil-DNA glycosylase activity. Here we identify a new class of DNA glycosylases in the UDG superfamily that lacks UDG activity. Instead, these enzymes act as hypoxanthine-DNA glycosylases in vitro and in vivo. Molecular modeling and structure-guided mutational analysis allowed us to identify a unique catalytic center in this class of DNA glycosylases. Based on unprecedented biochemical properties and phylogenetic analysis, we propose this new class of DNA repair glycosylases that exists in bacteria, archaea, and eukaryotes as family 6 and designate it as the hypoxanthine-DNA glycosylase family. This study demonstrates the structural evolvability that underlies substrate specificity and catalytic flexibility in the evolution of enzymatic function.

FIGURE 1.

Sequence motifs and deaminated base repair activity of Mba DNA glycosylase. A, sequence motifs of the UDG superfamily. GenBank accession numbers are shown after the species names. Family 6 (HDG), Mba, YP_304295.1. Family 1 (UNG), E. coli (Eco), NP_289138. Family 2 (MUG/TDG), Homo sapiens (Hsa), NP_003202. Family 3 (SMUG1), Geobacter metallireducens (Gme) GS-15, YP_383069. Family 4 (UDGa), Pyrobaculum aerophilum (Pae) str. IM2, NP_558739.1. Family 5 (UDGb), P. aerophilum (Pae) str. IM2, NP_559226. B, Deaminated base repair activity from Mba DNA glycosylase. DNA glycosylase activity assays were performed as described under “Experimental Procedures” with 100 nm WT Mba glycosylase protein and 10 nm oligonucleotide substrate. A control reaction was performed with E. coli UNG (lane 21). X, U, I, and O, xanthosine-, uridine-, inosine-, and oxanosine-containing DNA. Lane 1, C/X; lane 2, G/X; lane 3, A/X; lane 4, T/X; lane 5, single-stranded X; lane 6, C/U; lane 7, G/U; lane 8, A/U; lane 9, T/U; lane 10, single-stranded U; lane 11, C/I; lane 12, G/I; lane 13, A/I; lane 14, T/I; lane 15, single-stranded I; lane 16, C/O; lane 17, G/O; lane 18, A/O; lane 19, T/O; lane 20, single-stranded O; lane 21, G/U.

FIGURE 2.

Catalytic center and role of Mba hypoxanthine-DNA glycosylase activity in vivo. A, modeled structure of Mba DNA glycosylase. The apurinic/apyrimidinic site, Leu-22, Asp-25, Asn-39, Asp-74, Asp-86, and Asn-113 are shown in color. The conserved Asn-95 is also shown in color where the distance between the side chain oxygen (O^δ) and the anomeric carbon is 17.70 Å. B, hypoxanthine-DNA glycosylase activity of N39A, N39D, and N39Q mutants. DNA glycosylase activity assays were performed as described under “Experimental Procedures” with 100 nm WT Mba glycosylase protein and 10 nm oligonucleotide substrate. Lane 1, C/I; lane 2, G/I; lane 3, A/I; lane 4, T/I; lane 5, single-stranded I. C, role of Mba DNA glycosylase in vivo. The numbers of revertants were scored on minimal lactose plates after incubation at 37 °C for 4 days. The data are the means ± S.D. of three independent experiments. S.D. is indicated by error bars. Open bar, N39A mutant of Mba DNA glycosylase; hatched bar, WT Mba DNA glycosylase.

FIGURE 3.

Phylogenetic analysis of UDG superfamily. The phylogenetic analysis was performed using the neighbor-joining method in MEGA 5 (27). GenBank accession numbers are shown after the species names. Family 1 (UDG), Deinococcus radiodurans (Dra) R1, NP_294412; E. coli (Eco), NP_289138; H. sapiens (Hsa), NP_003353; and Mycobacterium tuberculosis (Mtu) H37Rv, CAB05436.1. Family 2 (MUG/TDG), Aspergillus clavatus (Acl) NRRL 1, XP_001268386.1; D. radiodurans (Dra) R1, NP_294438; E. coli (Eco), P0A9H1; H. sapiens (Hsa), NP_003202; and S. pombe (Spo), O59825. Family 3 (SMUG1), Drosophila melanogaster (Dme), NP_650609.1; G. metallireducens (Gme) GS-15, YP_383069; H. sapiens (Hsa), NP_055126; Strongylocentrotus purpuratus (Spu), XP_782746.1; and Xenopus laevis (Xla), AAD17300. Family 4 (UDGa), Nitrosospira multiformis (Nmu), YP_412806; P. aerophilum (Pae) str. IM2, NP_558739.1; Thermotoga maritima (Tma) MSB8, NP_228321.1; and Thermus thermophilus (Tth) HB27, YP_004341.1. Family 5 (UDGb), M. tuberculosis (Mtu) H37Rv, P64785 (Rv1259); P. aerophilum (Pae) str. IM2, NP_559226; Streptomyces coelicolor (Sco) A3(2), NP_626251.1; and T. thermophilus (Tth) HB27, YP_004757.1. Family 6 (HDG), Burkholderia phymatum (Bph) STM815, YP_001858334.1; Candidatus Methanoregula boonei (Cme) 6A8, YP_001405361.1; Entamoeba histolytica (Ehi) HM-1:IMSS, XP_655177.1; Mba str. Fusaro, YP_304295.1; M. hungatei (Mhu) JF-1, YP_502486.1; Polaribacter sp. (Psp) MED152, ZP_05108206.1; and Ricinus communis (Rco), XP_002536323.1.