title: Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase beta.
abstract: We previously reported the mode of inhibition of DNA polymerase beta (pol. beta) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. beta interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1H-15N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA.
authors.affiliation: Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba-ken 278-8510, Japan.
authors.forename: Shizuka
authors.initials: S
authors.lastname: Murakami
authors.forename: Shinji
authors.initials: S
authors.lastname: Kamisuki
authors.forename: Kei-ichi
authors.initials: K
authors.lastname: Takata
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Kasai
authors.forename: Seisuke
authors.initials: S
authors.lastname: Kimura
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Mizushina
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ohta
authors.forename: Fumio
authors.initials: F
authors.lastname: Sugawara
authors.forename: Kengo
authors.initials: K
authors.lastname: Sakaguchi
mesh_terms: D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D000818:Animals; D019951:DNA Polymerase beta / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry*; D008095:Lithocholic Acid / Q000737:chemistry*; D008958:Models, Molecular; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D000596:Amino Acids; D004791:Enzyme Inhibitors; D005229:Fatty Acids, Monounsaturated; D010446:Peptide Fragments; D008095:Lithocholic Acid; C013147:nervonic acid; D019951:DNA Polymerase beta