The inhibitory action of pyrrolidine alkaloid, 1,4-dideoxy-1,4-imino-D-ribitol, on eukaryotic DNA polymerases.

The pyrrolidine alkaloids mimicking the structures of pentose with nitrogen in the ring are known to be inhibitors of glycosidases. We report here that a compound belonging to this category is an inhibitor of eukaryotic DNA polymerases. Among the eight naturally occurring pyrrolidine alkaloids we tested, only one compound, 1,4-dideoxy-1,4-imino-D-ribitol (DRB), which was purified from the mulberry tree (Morus alba), strongly inhibited the activities of eukaryotic DNA polymerases with IC50 values of 21-35 microM, and had almost no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase, and bovine deoxyribonuclease I. Kinetic studies showed that inhibition of both DNA polymerases alpha and beta by DRB was competitive with respect to dNTP substrate. Whereas DNA polymerase alpha inhibition was noncompetitive with the template-primer, the inhibition of DNA polymerase beta was found to be competitive with the template-primer. The K(i) values of DNA polymerases alpha and beta for the template-primer were smaller than those for dNTP substrate. Therefore, the affinity of DRB was suggested to be higher at the template-primer binding site than at the dNTP substrate-binding site, although DRB is an analogue of deoxyribose consisting of dNTP. Computational analyses of the eight pyrrolidine alkaloids revealed a remarkable difference in the distribution of positive and negative electrostatic charges on the surface of molecules. The relationship between the structure of DRB and the inhibition of eukaryotic DNA polymerases is discussed.

Stimulation of human DNA polymerase epsilon by MDM2.

The human DNA polymerase epsilon catalytic subunit consists of a 140-kDa N-terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2. We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase epsilon up to 10- and 40-fold, respectively, but not those of DNA polymerase beta or Klenow fragment of E.coli DNA polymerase I. Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities. The stimulation depended upon the interaction of the N-terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N-terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase epsilon by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation. Since DNA polymerase epsilon appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase epsilon might be part of a reconfiguration process that allows DNA polymerase epsilon to associate with repair/recombination proteins in response to DNA damage.

A sulphoquinovosyl diacylglycerol is a DNA polymerase epsilon inhibitor.

Sulphoquinovosyl diacylglycerol (SQDG) was reported as a selective inhibitor of eukaryotic DNA polymerases alpha and beta [Hanashima, Mizushina, Ohta, Yamazaki, Sugawara and Sakaguchi (2000) Jpn. J. Cancer Res. 91, 1073-1083] and an immunosuppressive agent [Matsumoto, Sahara, Fujita, Shimozawa, Takenouchi, Torigoe, Hanashima, Yamazaki, Takahashi, Sugawara et al. (2002) Transplantation 74, 261-267]. The purpose of this paper is to elucidate the biochemical properties of the inhibition more precisely. As expected, SQDG could inhibit the activities of mammalian DNA polymerases such as alpha, delta, eta and kappa in vitro in the range of 2-5 micro M, and beta and lambda in vitro in the range of 20-45 micro M. However, SQDG could inhibit only mammalian DNA polymerases epsilon (pol epsilon) activity at less than 0.04 micro M. SQDG bound more tightly to mammalian pol epsilon than the other mammalian polymerases tested. Moreover, SQDG could inhibit the activities of all the polymerases from animals such as fish and insect, but not of the polymerases from plant and prokaryotes. SQDG should, therefore, be called a mammalian pol epsilon-specific inhibitor or animal polymerase-specific inhibitor. To our knowledge, this represents the first report about an inhibitor specific to mammalian pol epsilon.

Petasiphenol: a DNA polymerase lambda inhibitor.

Petasiphenol, a bio-antimutagen isolated from a Japanese vegetable, Petasites japonicus, selectively inhibits the activities of mammalian DNA polymerase lambda (pol lambda) in vitro. The compound did not influence the activities of replicative DNA polymerases such as alpha, delta, and epsilon but also showed no effect even on the pol beta activity, the three-dimensional structure of which is thought to be highly similar to pol lambda. The inhibitory effect of petasiphenol on intact pol lambda including the BRCA1 C-terminus (BRCT) domain was dose-dependent, and 50% inhibition was observed at a concentration of 7.8 microM. The petasiphenol-induced inhibition of the pol lambda activity was noncompetitive with respect to both the DNA template-primer and the dNTP substrate. Petasiphenol did not only inhibit the activity of the truncated pol lambda including the pol beta-like core, in which the BRCT motif was deleted in its N-terminal region. BIAcore analysis demonstrated that petasiphenol bound selectively to the N-terminal domain of pol lambda but did not bind to the C-terminal region. On the basis of these results, the pol lambda inhibitory mechanism of petasiphenol is discussed.

Vitamin A-related compounds, all-trans retinal and retinoic acids, selectively inhibit activities of mammalian replicative DNA polymerases.

Retinoic acids, vitamin A-related compounds, are known to be inhibitors of telomerase. We found that fucoxanthin from the sea alga Petalonia bingamiae is a potent inhibitor of mammalian replicative DNA polymerases (i.e., pol alpha, delta and epsilon). Since fucoxanthin is a carotenoid (provitamin A-related) compound, we characterized the biochemical modes of vitamin A-related compounds including vitamin A and provitamin A in this report. Subsequently, we found that fucoxanthin, all-trans retinal (RAL, vitamin A aldehyde) and all-trans retinoic acid (RA, vitamin A acid) inhibited the activities of replicative DNA polymerases with IC(50) values of 18-190, 14-17 and 8-30 microM, respectively. On the other hand, all-trans retinol (vitamin A) did not influence any of the DNA polymerase activities. RA inhibited not only the activities of pol alpha, delta and epsilon with IC(50) values of 30, 28 and 8 microM, respectively, but of pol beta with an IC(50) value of 27 microM. The tested vitamin A-related compounds did not influence the activities of DNA polymerases from a higher plant, cauliflower, prokaryotic DNA polymerases, or DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. RAL and RA should be called selective inhibitors of mammalian DNA polymerases including telomerase, and RAL was a specific inhibitor of mammalian replicative DNA polymerases. As expected from these results in vitro, some of them could prevent the growth of NUGC-3 human gastric cancer cells, and especially RAL was a potent antineoplastic agent with an LD(50) value of 19 microM. The cells were halted at G1 phase in the cell cycle by RAL.

A plant phytotoxin, solanapyrone A, is an inhibitor of DNA polymerase beta and lambda.

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.