On the fidelity of deoxyribonucleic acid synthesis directed by chromatin-associated deoxyribonucleic acid polymerase .beta.

Abstract

Accuracy of poly[d(A-T)] synthesis catalyzed by chromatin-bound DNA polymerase .beta. was measured with several cell types [including mouse and human cells]. 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 MW poly[d(A-T)] by gel filtration. The efficacy of DNA removal by this procedure was validated by CsCl2 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 polymerase 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, the fidelity of this reaction is exceptionally low; approximately 1 dGTP was incorporated for every thousand complementary nulceotides polymerized.