We have investigated the properties of two possible DNA polymerase accessory proteins in mammalian cells: a DNA-dependent ATPase endowed with some 'helicase' activity; and a new class of DNA-binding proteins (MR 22,000-27,000 daltons). Both factors are able to stimulate specifically the DNA polymerase ? by at least a 10-fold factor on poly d(A-T) and on other appropriate primed templates. Their action is additive and, under certain conditions, slightly synergistic, indicating a possible functional interaction. Both factors act by destabilizing the double helix, thus easing the advancement of the DNA polymerase ? on the template. We have developed purification conditions of high-salt nuclear extract from HeLa cells which allow the separation of 'alkaline' DNases from DNA polymerases. This fraction, on Sephadex G-100, was divided into three main peaks: the first (MR about 100,000 daltons) contains a single-stranded 3' -> 5' exonuclease, the second (MR about 50,000 daltons) is a mixture of DNase VI (single-stranded endo) and of a 3' -> 5' exonuclease working equally well on single-stranded and duplex DNA; this latter activity is similar in some respects to DNase III, purified from rabbit bone marrow. The third peak contains 5' -> 3' single-stranded exonuclease (MR about 30,000 acting on UV-irradiated substrate, whose reaction products are short oligonucleotides containing pyrimidine dimers.
Enzymes of DNA metabolism in animal cells
Cobianchi F;
1982
Abstract
We have investigated the properties of two possible DNA polymerase accessory proteins in mammalian cells: a DNA-dependent ATPase endowed with some 'helicase' activity; and a new class of DNA-binding proteins (MR 22,000-27,000 daltons). Both factors are able to stimulate specifically the DNA polymerase ? by at least a 10-fold factor on poly d(A-T) and on other appropriate primed templates. Their action is additive and, under certain conditions, slightly synergistic, indicating a possible functional interaction. Both factors act by destabilizing the double helix, thus easing the advancement of the DNA polymerase ? on the template. We have developed purification conditions of high-salt nuclear extract from HeLa cells which allow the separation of 'alkaline' DNases from DNA polymerases. This fraction, on Sephadex G-100, was divided into three main peaks: the first (MR about 100,000 daltons) contains a single-stranded 3' -> 5' exonuclease, the second (MR about 50,000 daltons) is a mixture of DNase VI (single-stranded endo) and of a 3' -> 5' exonuclease working equally well on single-stranded and duplex DNA; this latter activity is similar in some respects to DNase III, purified from rabbit bone marrow. The third peak contains 5' -> 3' single-stranded exonuclease (MR about 30,000 acting on UV-irradiated substrate, whose reaction products are short oligonucleotides containing pyrimidine dimers.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.