Mode Analysis of a Fatty Acid Molecule Binding to the N-terminal 8-kDa Domain of DNA Polymerase β: A 1:1 COMPLEX AND BINDING SURFACE

Yoshiyuki Mizushina,Tadayasu Ohkubo,Takayasu Date,Toyofumi Yamaguchi,Mineo Saneyoshi,Fumio Sugawara,Kengo Sakaguchi

doi:10.1074/jbc.274.36.25599

Yoshiyuki Mizushina, Tadayasu Ohkubo + Show 5 more

Open Access

https://doi.org/10.1074/jbc.274.36.25599

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Abstract

We reported previously that long-chain fatty acids are potent inhibitors of mammalian DNA polymerase beta. 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 Omega-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.

Highlights

We reported previously that long-chain fatty acids strongly inhibited the activities of mammalian DNA polymerase ␣1 and DNA polymerase ␤ 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]
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 ␤
Fatty acids in which they are of the trans-configuration have much weaker inhibitory effects on especially pol ␤, and the fatty acids in which the carboxyl end is chemically modified can lose the inhibitory effect

Summary

Introduction

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]. We analyzed the structural interactions of C18 and C24 fatty acids with pol ␤, especially the N-terminal 8-kDa domain, in cross-linking studies using a 5Ј-end-labeled photoprobe (dT14D) instead of template-primer DNA and the binding surface of the 8-kDa domain in contact with the fatty acids by NMR.

Results

Conclusion