We have cloned the human mutY gene (hMYH) from both genomic and cDNA libraries. The human gene contains 15 introns and is 7.1 kb long. The 16 exons encode a protein of 535 amino acids that displays 41% identity to the Escherichia coli protein, which provides an important function in the repair of oxidative damage to DNA and helps to prevent mutations from oxidative lesions. The human mutY gene maps on the short arm of chromosome 1, between p32.1 and p34.3.The study of bacterial and yeast repair genes and their involvement in preventing mutations has facilitated the understanding of mutagenesis and repair in humans. A stunning example of this is the recent demonstration that inherited susceptibility to hereditary nonpolyposis colon cancer (HNPCC) is due to the inheritance of a defective copy of one of the human homologs of the bacterial mismatch repair system (4,6,8,19). Defects in either the Escherichia coli mutH, -L, or -S or the uvrD gene lead to a nonfunctional mismatch repair system and an increased rate of spontaneous mutations, the "mutator" phenotype (16). In humans, a mutator effect, easily detectable by observing instability of microsatellite repeats (8, 9), is also seen to result from defects in the mismatch repair system. This has led to renewed interest in characterizing mutator bacteria that might define additional repair systems and in finding their counterparts in humans.We have described two mutator genes in E. coli, the mutY and the mutM genes (5, 17), which work together to prevent mutations from certain types of oxidative damage, dealing in particular with the oxidized guanine lesion 8-oxodG (13). Figure 1 (see also reference 14) summarizes the concerted action of the enzymes encoded by these two genes, both of which are glycosylases. The MutM protein removes 8-oxodG from the DNA, and the resulting a purinic site is repaired to restore the GC base pair. Some lesions are not repaired before replication, which results in a GC-to-TA transversion at the next round of replication because polymerases involved in DNA replication incorporate A across from 8-oxoG between 5-fold and 200-fold more frequently than they incorporate C (22). However, the MutY protein removes the A across from 8-oxodG and repair synthesis restores a C most of the time (since polymerases involved in repair insert C in preference to A across from 8-oxoG [22]), allowing the MutM protein another opportunity to repair the lesion. In accordance with this, mutators lacking either the MutM or the MutY protein have an increase specifically in the GC-to-TA transversion (5, 17), and cells lacking both enzymes have an enormous increase in this base substitution (13). A third protein, the product of the mutT gene, prevents the incorporation of 8-oxodGTP by hydrolyzing the oxidized triphosphate back to the monophosphate (11), preventing AT-to-CG transversions.The human homolog of the E. coli mutT gene has been cloned and sequenced (20). Here we describe the characterization of the human homolog of the mutY gene (hMYH). The gene is 7.1 kb l...

Proteins containing the Nudix box "GX 5 EX 7 REUXE-EXGU" (where U is usually Leu, Val, or Ile) are Nudix hydrolases, which catalyze the hydrolysis of a variety of nucleoside diphosphate derivatives. Here we report cloning and characterization of a human cDNA encoding a novel nudix hydrolase NUDT5 for the hydrolysis of ADP-sugars. The deduced amino acid sequence of NUDT5 contains 219 amino acids, including a conserved Nudix box sequence. The recombinant NUDT5 was expressed in Escherichia coli and purified to near homogeneity. At the optimal pH of 7, the purified recombinant NUDT5 catalyzed hydrolysis of two major substrates ADP-ribose and ADP-mannose with K m values of 32 and 83 M, respectively; the V max for ADP-mannose was about 1.5 times that with ADP-ribose. The murine NUDT5 homolog was also cloned and characterized. mNudT5 has 81% amino acid identity to NUDT5 with catalytic activities similar to NUDT5 under the optimal pH of 9. Both NUDT5 and mNudT5 transcripts were ubiquitously expressed in tissues analyzed with preferential abundance in liver. The genomic structures of both NUDT5 and mNudT5 were determined and located on human chromosome 10 and mouse chromosome 2, respectively. The role of NUDT5 in maintaining levels of free ADP-ribose in cells is discussed.

We have previously described the hMYH cDNA and genomic clones (M. M. Slupska et al., J. Bacteriol. 178:3885–3892, 1996). Here, we report that the enzyme expressed from an hMYH cDNA clone in Escherichia coli complements the mutator phenotype in a mutY mutant and can remove A from an A · 8-hydroxydeoxyguanine mismatch and to a lesser extent can remove A from an A · G mismatch in vitro.

This work presents the first example of an anti-mutator role of the sbcC,D genes, and defines a new gene (rmuC) involved in DNA recombination.