Accumulation of small fragments of DNA in isolated HeLa cell nuclei due to transient incorporation of dUMP.
[3H]dUMP was incorporated into DNA of isolated S-phase HeLa S3 cell nuclei during DNA synthesis. The incorporated radioactivity was made acid soluble during a chase with excess TTP. A partially purified DNA polymerase alpha incorporated [3H]dUMP into activated salmon sperm DNA. The incorporation rate was equal to the incorporation of [3H]TMP, and the radioactivity incorporated was not made acid soluble during a chase. The nuclei thus have the ability to remove misincorporated uracil. From cytosol we have partially purified an enzyme (80 times purification) that splits the N-glycosidic bond between uracil and deoxyribose in dUMP-containing DNA. This uracil-N-glycosidase has a molecular weight of about 50 000. It does not accept dUTP or RNA as substrates. Pulse labelling of isolated nuclei with radioactive deoxyribonucleoside triphosphates in the presence of dUTP lead to a large accumulation of label in small DNA fragments. The size of these fragments was about 80 nucleotides in a 60 s pulse and no increase in size was observed with increasing pulse length. The corresponding value for control experiments with no dUTP, was 200 nucleotides and the fragments increased in size with increasing pulse length. About 90% of the radioactivity was found in the small fragments after a 3 min pulse when the concentration of dUTP in the test mixture was 100 micrometer and no exogenous TTP was present. In control experiments with no dUTP present, only 14% of the radioactivity was found in small DNA pieces. When test mixture containing dUTP was preincubated with cytosol for 60 s before adding the isolated nuclei, the small fragments increased in size to that of DNA fragments found in control incubations; also the relative amount of label bound to the fragments returned to the levels found in the controls. Increasing the TTP concentration from 5 micrometer to 1.88 mM in the absence of exogenous dUTP had no effect on the size of the DNA fragments.
D002453:Cell Cycle; D002467:Cell Nucleus / Q000378:metabolism*; D004257:DNA Polymerase II / Q000378:metabolism; D004273:DNA, Neoplasm / Q000096:biosynthesis*; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism*; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism
D004273:DNA, Neoplasm; D003856:Deoxyuracil Nucleotides; C007267:2'-deoxyuridylic acid; D004257:DNA Polymerase II; D009699:N-Glycosyl Hydrolases