Inhibition of chicken myeloblastosis RNA polymerase II activity by adriamycin.

In vitro RNA synthesis by isolated RNA polymerase II of chicken myeloblastosis cells was shown to be highly sensitive to adriamycin inhibition. The template activity of the single-stranded DNA, purified by chromatography of denatured calf thymus DNA through hydroxylapatite columns, was found to be equally as sensitive to the inhibition as denatured calf thymus DNA. However, contrary to denatured DNA, the single-stranded DNA thus purified showed no significant binding to adriamycin as analyzed by cosedimentation of the drug and DNA through a sucrose gradient. This indicated that inhibition of RNA synthesis on a single-stranded DNA template might involve a mechanism other than DNA intercalation. Kinetic studies of the inhibition showed that the inhibition of RNA synthesis by adriamycin could not be reversed by increasing the concentrations of RNA polymerase and four nucleoside triphosphates, but it could be reversed by increasing DNA concentrations. Analysis of the size of RNA synthesized indicated that the ultimate size of the product RNA was not altered by adriamycin, suggesting that the drug may inhibit RNA synthesis by reducing RNA chain initiation.     

A new DNA-dependent ATPase which stimulates yeast DNA polymerase I and has DNA-unwinding activity

Two forms of DNA-dependent ATPase activity were previously purified from the yeast Saccharomyces cerevisiae and characterized (Plevani, P., Badaracco, G., and Chang, L. M. S. (1980) J. Biol. Chem. 255, 4957-4963). Here, an additional DNA-dependent ATPase (ATPase III) has been purified from S. cerevisiae to near homogeneity. This ATPase differs from those described previously in its chromatographic properties, molecular weight, reaction properties and immunological relatedness. Its molecular weight is about 63,000 in the presence of sodium dodecyl sulfate. It hydrolyzes ATP to ADP and orthophosphate in the presence of DNA as an effector. In addition, yeast DNA polymerase I, which is a true DNA replicase of yeast, is stimulated severalfold by this ATPase. Neither yeast DNA polymerase II nor prokaryotic DNA polymerases are stimulated. This stimulation is intrinsic to the ATPase activity, since both activities copurified in the last four steps of purification, showed the same heat stability and showed dependence on and hydrolysis of ATP. The ATPase III preparation also contains a DNA-unwinding (DNA helicase) activity, which unwinds double-stranded DNA in the presence of ATP. In the S. cerevisiae radiation-sensitive mutant rad3, no significant ATPase III activity could be detected, suggesting that the RAD3 gene, which codes for a different polypeptide, regulates the expression of ATPase III activity.     

Acquisition of new DNA sequences after infection of chicken cells with avian myeloblastosis virus

DNA-RNA hybridization studies between 70S RNA from avian myeloblastosis virus (AMV) and an excess of DNA from (i) AMV-induced leukemic chicken myeloblasts or (ii) a mixture of normal and of congenitally infected K-137 chicken embryos producing avian leukosis viruses revealed the presence of fast- and slow-hybridizing virus-specific DNA sequences. However, the leukemic cells contained twice the level of AMV-specific DNA sequences observed in normal chicken embryonic cells. The fast-reacting sequences were two to three times more numerous in leukemic DNA than in DNA from the mixed embryos. The slow-reacting sequences had a reiteration frequency of approximately 9 and 6, in the two respective systems. Both the fast- and the slow-reacting DNA sequences in leukemic cells exhibited a higher T_m (2 C) than the respective DNA sequences in normal cells. In normal and leukemic cells the slow hybrid sequences appeared to have a T_m which was 2 C higher than that of the fast hybrid sequences. Individual non-virus-producing chicken embryos, either group-specific antigen positive or negative, contained 40 to 100 copies of the fast sequences and 2 to 6 copies of the slowly hybridizing sequences per cell genome. Normal rat cells did not contain DNA that hybridized with AMV RNA, whereas non-virus-producing rat cells transformed by B-77 avian sarcoma virus contained only the slowly reacting sequences. The results demonstrate that leukemic cells transformed by AMV contain new AMV-specific DNA sequences which were not present before infection.     

Carboxy terminal region of a chloroplast DNA polymerase accessory factor stimulates DNA polymerase activity

p43, a glycoprotein of pea chloroplast (ct), acts as an accessory protein of pea chloroplast DNA polymerase. p43 binds to DNA, binds to ct-DNA polymerase and stimulates the ct-DNA polymerase activity. In the work presented here, the C-terminal domain of p43 (p22) has been overexpressed in E. coli. South Western analysis reveals that the recombinant p22 lacks in DNA binding activity. However, the recombinant p22 can form complex with the pea ct-DNA polymerase quite efficiently and stimulates the DNA polymerase activity to a greater extent than the native p43. Thus the DNA binding domain of p43 appears to be spatially separate from the domain responsible for the DNA polymerase accessory activity. The DNA binding domain is also highly O-glycosylated and loss of glycosylation of p43 leads to enhanced DNA binding as well as repression of ct-DNA polymerase activity. These findings allow us to propose a model to explain how glycosylation of p43 helps ct-DNA polymerase latch onto the DNA template for enhanced processivity. The predictive components of the model have been discussed.     

Structural design of a eukaryotic DNA repair polymerase: DNA polymerase beta

DNA polymerase beta, the smallest eukaryotic DNA polymerase, is designed to synthesize DNA in short DNA gaps during DNA repair. It is composed of two specialized domains that contribute essential enzymatic activities to base excision repair (BER). Its amino-terminal domain possesses a lyase activity necessary to remove the 5'-deoxyribose phosphate (dRP) intermediate generated during BER. Removal of the dRP moiety is often the rate-limiting step during BER. Failure to remove this group may initiate alternate BER pathways. The larger polymerase domain has nucleotidyl transferase activity. This domain has a modular organization with sub-domains that bind duplex DNA, catalytic metals, and the correct nucleoside triphosphate in a template-dependent manner. X-ray crystal structures of DNA polymerase beta, with and without bound substrates, has inferred that domain, sub-domain, and substrate conformational changes occur upon ligand binding. Many of these conformational changes are distinct from those observed in structures of other DNA polymerases. This review will examine the structural aspects of DNA polymerase beta that facilitate its role in BER.