Involvement of deoxyribonucleic acid polymerase beta in nuclear deoxyribonucleic acid synthesis.

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.     

Structure of Caulobacter deoxyribonucleic acid.

The deoxyribonucleic acid of the dimorphic bacterium Caulobacter crescentus contains a component that renatures with rapid, unimolecular kinetics. This component was present in both swarmer and stalked cells and exhibited the sensitivity to endonuclease S1 expected for hairpin loops. Double-stranded side branches between 100 and 600 nucleotide pairs in length were visible in electron micrographs of rapidly reassociating deoxyribonucleic acid isolated by hydroxyapatite chromatography. No extrachromosomal elements were found in spite of systematic attempts to detect their presence. These results indicate that the rapidly reassociating fraction derives from inverted repeat sequences within the chromosome and not from cross-links or plasmids. We estimate that there are approximately 350 inverted repeat regions per Caulobacter genome. The kinetic complexity of Caulobacter deoxyribonucleic acid, however, is no greater than that of other bacteria.     

Nuclear deoxyribonucleic acid polymerases of liver.

Hepatic nuclei that are isolated in aquenous solutions of low ionic strength or glycerol contain all or nearly all the nonmitochondrial DNA polymerase activity of the cell. The presence of polymerase activity in the cytoplasm is due to extraction of nuclear enzymes by buffer and inorganic salts. Even with low ionic strength solutions, some leaching of nuclear enzymes occurs if the concentration of liver in the homogenizing medium is greater than 10%. As defined by sucrose gradient analysis, the normal adult rat liver nucleus contains mainly or entirely a single species of DNA polymerase (3.2 S) whereas the regenerating nucleus after 70% hepatectomy has an additional enzyme (7.1 S). The total activity of regenerating nuclei is about twice the normal value. The increase resides in the 7.1 S activity. The 7.1 S DNA polymerase had been purified partially from regenerating liver nuclei (isolated in low ionic strength solutions) and cytosol (prepared under conditions of nuclear enzyme extraction). The properties of the activity from the two sources are indistinguishable. A mixture of albumin and spermidine enhances by several-fold the activities of the 3.2 S and 7.1 S DNA polymerases. In the presence of spermidine, but not in its absence, the activity of the 7.1 S DNA polymerase is strictly proportional to the amount of the enzyme preparation.