A Plant Phytotoxin, Solanapyrone A, Is an Inhibitor of DNA Polymerase β and λ

Yoshiyuki Mizushina,Shinji Kamisuki,Nobuyuki Kasai,Noriko Shimazaki,Masaharu Takemura,Hitomi Asahara,Stuart Linn,Shonen Yoshida,Akio Matsukage,Osamu Koiwai,Fumio Sugawara,Hiromi Yoshida,Kengo Sakaguchi

doi:10.1074/jbc.m105144200

Abstract

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase beta and lambda (pol beta and lambda) in vitro. The IC50 values of the compound were 30 microm for pol beta and 37 microm for pol lambda. Because pol beta and lambda are in a family and their three-dimensional structures are thought to be highly similar to each other, we used pol beta to analyze the biochemical relationship with solanapyrone A. On pol beta, solanapyrone A antagonistically competed with both the DNA template and the nucleotide substrate. BIAcore analysis demonstrated that solanapyrone A bound selectively to the N-terminal 8-kDa domain of pol beta. This domain is known to bind single-stranded DNA, provide 5'-phosphate recognition of gapped DNA, and cleave the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity) including 5'-deoxyribose phosphate lyase activity. Solanapyrone A inhibited the single-stranded DNA-binding activity but did not influence the activities of the 5'-phosphate recognition in gapped DNA structures and the AP lyase. Based on these results, the inhibitory mechanism of solanapyrone A is discussed.

Highlights

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase ␤ and ␭ in vitro
Because at least pol ␤ is an essential enzyme for nucleotide excision repair [1, 2], the plant phytotoxin may lead to blockage of the DNA repair systems of rescue cancer cells under clinical radiation-therapy or chemotherapy
Negative FABHR (Fast Atom Bombardment High Resolution) mass and [1H]NMR, [13C]NMR, and DEPT (Distortionless Enhancement by Polarization Transfer) NMR spectroscopic analyses indicated that the inhibitor was found to be an agent known as solanapyrone A previously reported as a phytotoxin isolated together with other toxic metabolites from A. solani, the causal fungus of early potato blight [28]

Summary

Introduction

Solanapyrone A, a phytotoxin and enzyme inhibitor isolated from a fungus (SUT 01B1-2) selectively inhibits the activities of mammalian DNA polymerase ␤ and ␭ (pol ␤ and ␭) in vitro. The biochemical mode of action is still unknown, there is a report on the detection of the enzymatic activity, isolation, and characterization of an enzyme catalyzing a Diels-Alder reaction [29] This compound was found to bind directly to the 8-kDa domain of pol ␤, but not to the 31-kDa domain, and in the three functional activities of the 8-kDa domain (i.e. ssDNA binding, 5Ј-phosphate recognition in gapped DNA, and dRP lyase activity using intact AP site containing DNA substrate) to inhibit only the ssDNA-binding activity. Based on these results, the inhibitory action of solanapyrone A and its relation to the enzyme structure of the 8-kDa domain of pol ␤ is discussed under “Discussion.”

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Conclusion