Specific amino acid residues are involved in substrate discrimination and template binding of human REV1 protein.
REV1 is a member of the Y-family DNA polymerases, but is atypical in utilizing only dCTP with a preference for guanine (G) as the template. Crystallography of the REV1-DNA-dCTP ternary complex has revealed a unique mechanism by which template G is evicted from the DNA helix and incoming dCTP is recognized by an arginine residue in an alpha-loop, termed the N-digit. To better understand functions of its individual amino acid residues, we made a series of mutant human REV1 proteins. We found that R357 and L358 play vital roles in template binding. Furthermore, extensive mutation analysis revealed a novel function of R357 for substrate discrimination, in addition to previously proposed specific interaction with incoming dCTP. We found that the binding pocket for dCTP of REV1 has also significant but latent affinity for dGTP. The results suggest that the positive charge on R357 could prevent interaction with dGTP. We propose that both direct and indirect mechanisms mediated by R357 ensure specificity for dCTP.
Piao
Jinlian
J
Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.
Masuda
Yuji
Y
Kamiya
Kenji
K
D000595:Amino Acid Sequence / Q000235:genetics; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D003845:Deoxycytosine Nucleotides / Q000378:metabolism*; D006801:Humans; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic; D000099270:Y-Family DNA Polymerases
D001120:Arginine; D004247:DNA; D003845:Deoxycytosine Nucleotides; D007930:Leucine; D009687:Nuclear Proteins; D009713:Nucleotidyltransferases; C024107:2'-deoxycytidine 5'-triphosphate; C429004:REV1 protein, human; D000099270:Y-Family DNA Polymerases