Ntg2p, a Saccharomyces cerevisiae DNA N-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidative DNA damage, interacts with the DNA mismatch repair protein Mlh1p. Identification of a Mlh1p binding motif.
Ntg2p is a DNA N-glycosylase/apurinic or apyrimidinic lyase involved in base excision repair of oxidatively damaged DNA in Saccharomyces cerevisiae. Using a yeast two-hybrid screen and a GST in vitro transcription and translation assay, the mismatch repair (MMR) protein Mlh1p was demonstrated to interact physically with Ntg2p. The Mlh1p binding site maps to amino acids residues 15-40 of Ntg2p. The Ntg2p binding site is localized in the C-terminal end (483-769) of Mlh1p. Overproduction of Ntg2p results in a mutator phenotype with enhanced frameshift reversion frequency, suggesting partial inhibition of the MMR pathway. In contrast, inactivation of NTG2 does not enhance mutagenesis, indicating that Ntg2p is not required for MMR. Site-directed mutagenesis of the Mlh1p binding domain of Ntg2p revealed three amino acids (Ser(24), Tyr(26), Phe(27)) that are absolutely required for Ntg2p-Mlh1p interaction. These residues are part of a motif found in Ntg2p (Arg(23)-Ser(24)-Lys(25)-Tyr(26)-Phe(27)), Exo1p (Arg(444)-Ser(445)-Lys(446)-Phe(447)-Phe(448)), and Sgs1p (Lys(1383)-Ser(1384)-Lys(1385)-Phe(1386)-Phe(1387)). In these three proteins, the motif is part of the domain that interacts with the C-terminal end of Mlh1p. Furthermore, S445A, F447A, and F448A mutants of Exo1p do not bind Mlh1p, but the wild type Exo1p does. Therefore, we propose that the R/K-S-R/K-Y/F-Y/F sequence could define a Mhl1 binding motif. The results also suggest that base excision repair and MMR can cooperate to prevent deleterious effects of oxidative DNA damage.
Gellon
Lionel
L
Commissariat à l'Energie Atomique, Département de Radiobiologie et Radiopathologie, UMR217 CNRS/CEA Radiobiologie Moléculaire et Cellulaire, Fontenay aux Roses 92265, France.
Werner
Michel
M
Boiteux
Serge
S
D048868:Adaptor Proteins, Signal Transducing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004249:DNA Damage; D017931:DNA Primers; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D018384:Oxidative Stress; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; D005656:Fungal Proteins; C088859:MLH1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D009699:N-Glycosyl Hydrolases; D000070957:MutL Protein Homolog 1; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C116827:NTG2 protein, S cerevisiae