Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA.

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).     

Characterization of DNA synthesis and DNA-dependent ATPase activity at a restrictive temperature in temperature-sensitive tsFT848 cells with thermolabile DNA helicase B.

A temperature-sensitive mutant defective in DNA replication, tsFT848, was isolated from the mouse mammary carcinoma cell line FM3A. In mutant cells, the DNA-dependent ATPase activity of DNA helicase B, which is a major DNA-dependent ATPase in wild-type cells, decreased at the nonpermissive temperature of 39 degrees C. DNA synthesis in tsFT848 cells at the nonpermissive temperature was analyzed in detail. DNA synthesis measured by incorporation of [3H]thymidine decreased to about 50% and less than 10% of the initial level at 8 and 12 h, respectively. The decrease in the level of thymidine incorporation correlated with a decrease in the number of silver grains in individual nuclei but not with the number of cells with labeled nuclei. DNA fiber autoradiography revealed that the DNA chain elongation rate did not decrease even after an incubation for 10 h at 39 degrees C, suggesting that initiation of DNA replication at the origin of replicons is impaired in the mutant cells. The decrease in DNA-synthesizing ability coincided with a decrease in the level of the DNA-dependent ATPase activity of DNA helicase B. Partially purified DNA helicase B from tsFT848 cells was more heat sensitive than that from wild-type cells. Inactivation of DNA-dependent ATPase activity of DNA helicase B from mutant cells was considerably reduced by adding DNA to the medium used for preincubation, indicating that the DNA helicase of mutant cells is stabilized by binding to DNA.     

Synthesis and evaluation of AKR1C inhibitory properties of A-ring halogenated oestrone derivatives

Enzymes AKR1C regulate the action of oestrogens, androgens, and progesterone at the pre-receptor level and are also associated with chemo-resistance. The activities of these oestrone halides were investigated on recombinant AKR1C enzymes. The oestrone halides with halogen atoms at both C-2 and C-4 positions (13β-, 13α-methyl-17-keto halogen derivatives) were the most potent inhibitors of AKR1C1. The lowest IC₅₀ values were for the 13α-epimers 2_2I,4Br and 2_2I,4Cl (IC₅₀, 0.7 μM, 0.8 μM, respectively), both of which selectively inhibited the AKR1C1 isoform. The 13α-methyl-17-keto halogen derivatives 2_2Br and 2_4Cl were the most potent inhibitors of AKR1C2 (IC₅₀, 1.5 μM, 1.8 μM, respectively), with high selectivity for the AKR1C2 isoform. Compound 1_2Cl,4Cl showed the best AKR1C3 inhibition, and it also inhibited AKR1C1 (Ki: AKR1C1, 0.69 μM; AKR1C3, 1.43 μM). These data show that halogenated derivatives of oestrone represent a new class of potent and selective AKR1C inhibitors as lead compounds for further optimisations.     

Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development

Defects in the human XPV/POLH gene result in the variant form of the disease xeroderma pigmentosum (XP-V). The gene encodes DNA polymerase eta (Poleta), which catalyzes translesion synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers (CPDs) and other lesions. To further understand the roles of Poleta in multicellular organisms, we analyzed phenotypes caused by suppression of Caenorhabditis elegans POLH (Ce-POLH) by RNA interference (RNAi). F1 and F2 progeny from worms treated by Ce-POLH-specific RNAi grew normally, but F1 eggs laid by worms treated by RNAi against Ce-POLD, which encodes Poldelta did not hatch. These results suggest that Poldelta but not Poleta is essential for C. elegans embryogenesis. Poleta-targeted embryos UV-irradiated after egg laying were only moderately sensitive. In contrast, Poleta-targeted embryos UV-irradiated prior to egg laying exhibited severe sensitivity, indicating that Poleta contributes significantly to damage tolerance in C. elegans in early embryogenesis but only modestly at later stages. As early embryogenesis is characterized by high levels of DNA replication, Poleta may confer UV resistance in C. elegans, perhaps by catalyzing TLS in early embryogenesis.