On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase beta.
Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound deoxyribonucleic acid (DNA) polymerase beta was measured with several types. A new procedure was developed for the isolation of copied poly[d(A-T)] from chromatin DNA. This method involved in vitro copying of poly[d(A-T)] by native chromatin and subsequent selective fragmentation of chromatin by restriction nucleases, proteinase K, and heat denaturation. The fragmented natural DNA is then separated from the high molecular weight poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by cesium chloride gradient and nearest-neighbor analysis of the product of the reaction and by measurement of the fidelity of poly[d(A-T)] synthesis by Escherichia coli DNA Pol I contaminated with increasing amounts of DNA. Also, DNA polymerases dissociated from chromatin retain the same accuracy as that of native chromatin. Synthesis of poly[d(A-T)] by chromatin is catalyzed mainly by DNA polymerase-beta. By use of the described technique, we find that the fidelity of this reaction is exceptionally low; approximately one dGTP was incorporated for every thousand complementary nucleotides polymerized.
D000818:Animals; D002843:Chromatin / Q000201:enzymology*; D004256:DNA Polymerase I / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004262:DNA Restriction Enzymes; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008099:Liver / Q000201:enzymology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000201:enzymology; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains
D002843:Chromatin; D003609:Dactinomycin; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes