Mode Analysis of a Fatty Acid Molecule Binding to the N-terminal 8-kDa Domain of DNA Polymerase β

Abstract

We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase ␤. At present, based on information available from the NMR structure of the N-terminal 8-kDa domain, we examined the structural interaction with the 8-kDa domain using two species, C 18 -linoleic acid (LA) or C 24 -nervonic acid (NA). In the 8-kDa domain with LA or NA, the structure that forms the interaction interface included helix-1, helix-2, helix-4, the three turns (residues 1-13, 48 -51, and 79 -87) and residues adjacent to an ⍀-type loop connecting helix-1 and helix-2 of the same face. No significant shifts were observed for any of the residues on the opposite side of the 8-kDa domain. The NA interaction interface on the amino acid residues of the 8-kDa domain fragment was mostly the same as that of LA, except that the shifted cross-peaks of Leu-11 and Thr-79 were significantly changed between LA and NA. The 8-kDa domain bound to LA or NA as a 1:1 complex with a dissociation constant (K D ) of 1.02 or 2.64 mM, respectively.We reported previously that long-chain fatty acids strongly inhibited the activities of mammalian DNA polymerase ␣ (pol ␣) 1 and DNA polymerase ␤ (pol ␤) in vitro and plant DNA polymerases, albeit less potently, but that at the concentrations used, the fatty acids hardly influenced the activities of prokaryotic DNA polymerases or other DNA metabolic enzymes such as DNase I (1, 2). The most potent inhibitors were fatty acids, which have the following characteristics: hydrocarbon chain containing 18 or more carbons, a free carboxyl end, and the cis-configuration is preferred to the trans-configuration. Fatty acids in the trans-configuration have a much weaker inhibitory effect on pol ␤, and those in which the carboxyl end is chemically modified can lose the inhibitory effect on both pol ␣ and pol ␤. The mode of inhibition by longer chain fatty acids showed the same characteristics, except that the minimum inhibitory doses of these longer chain fatty acids were much lower (2, 3). Lineweaver-Burk plots of the fatty acids indicated that both the substrate (i.e. deoxynucleotide)-binding and the template DNA-binding sites of pol ␣ were nonantagonistically inhibited by the fatty acids, but they were effective as antagonists against the sites of pol ␤. For pol ␤, fatty acids acted by competing with not only the substrate but also the template-primer DNA. In screening inhibitors of eukaryotic DNA polymerase, we also found several natural compounds which inhibited pol ␤ in the same manner as fatty acids (4 -10).Pol ␤ is the smallest known DNA polymerase in animal cells with a molecular mass of 39 kDa, and its structure is highly conserved among mammals (11). This protein has a modular two-domain structure, with apparent flexibility within a protease-sensitive region between residues 82 and 86, which separates the two domains. Treatment with trypsin yields an N-terminal domain fragment (8 kDa), which retains binding affinity for single-stranded DNA (ssDNA), and a C-terminal domain fragment (31 kDa) ...

Structure‐Activity Relationship of a Novel Group of Mammalian DNA Polymerase Inhibitors, Synthetic Sulfoquinovosylacylglycerols

“…That is, each of the SQMG/SQDG appeared to inhibit these enzymes selectively, for example by binding to the hydrophobic region. Effects of synthetic SQMG/SQDG on cultured mammalian cells To investigate the tumoricidal effects of each of the SQMG/SQDG, the human stomach cancer cell line [12][13][14][15][16][17][18] . d) i.e., dTTP.…”

“…β by SQMG/SQDG, kinetic analyses were performed (Table II). Poly(dA)/oligo(dT) [12][13][14][15][16][17][18] (A/T=2/1) and dTTP were used as DNA template-primer and nucleotide substrate, respectively. Double reciprocal plots of the results showed that inhibition of pol.…”

“…4,5) One unit of DNA polymerase catalyzes the incorporation of 1 nmol of dTTP into synthetic template-primer (poly(dA)/oligo(dT) [12][13][14][15][16][17][18] , A/T=2/1) at 37°C in 60 min. The activities of RNA polymerase and polynucleotide kinase were measured in standard assays according to the manufacturer's specifications, as described by Nakayama and Saneyoshi 26) and Soltis and Uhlenbeck, 27) respectively.…”

“…1) In screening studies, we found many inhibitors of mammalian DNA polymerases. [4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] At present, we are engaged in analyzing the structure and function of the DNA polymerases using these inhibitors from two different viewpoints; to understand the precise role of each polymerase in vivo, and to develop a drug design strategy for cancer chemotherapy agents. Since DNA polymerases are essential enzymes for DNA replication and repair, and thus for cell division, inhibition of the enzymes will lead to killing of cells, especially under conditions of proliferation.…”

Antifungal properties of Japanese cedar essential oil from waste wood chips made from used sake barrels

“…The in vitro activities of DNA polymerase (8-10), DNA topoisomerase II (11), IMP dehydrogenase (12), polynucleotide kinase (8) and deoxyribonuclease (8) of T. rubrum mycelia extract were assayed at 37˚C as described previously (8)(9)(10)(11)(12). The enzymes were used at 0.05 units each.…”