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.     

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.     

Homology between the deoxyribonucleic acid of fertility factor P and Vibrio cholerae chromosomal deoxyribonucleic acid.

The deoxyribonucleic acid (DNA) of the Vibrio cholerae fertility factor P was isolated by the dye-buoyant density method and hybridized to V. cholerae chromosomal DNA. The DNA of this fertility plasmid had between 35 to 40% homology with the V. cholerae chromosomal DNA. Little or no homology was detected between the P factor DNA and DNA of the Escherichia coli sex factor F.     

Kinetic complexity of chloroplastal deoxyribonucleic acid and mitochondrial deoxyribonucleic acid from higher plants

1. Chloroplasts and mitochondria were isolated by aqueous and non-aqueous cell-fractionation techniques. In a variety of higher plants the mitochondrial DNA bands in a caesium chloride gradient at 1.706g.cm.(-3), whereas chloroplastal DNA has a buoyant density of 1.697g.cm.(-3). 2. In total cellular DNA of moderate molecular weight, the chloroplastal DNA is found within the Gaussian distribution of the nuclear DNA and is not resolved as a satellite. 3. Both chloroplastal DNA and mitochondrial DNA from lettuce renature rapidly. 4. The kinetic complexity of mitochondrial DNA is > 10(8) daltons. 5. Chloroplastal DNA is made up from fast and slow renaturing sequences with kinetic complexities of 3x10(6) and 1.2x10(8) daltons respectively. 6. From the discrepancy between analytical and kinetic complexity it is concluded that chloroplastal DNA is extensively reiterated.     

Denaturation of deoxyribonucleic acid in the cell

Summavy Isolated DNA loses in the smear its original ability to stain by methylene blue metachromatically, if it was denatured. Hepatocyte chromatin in a smear is stained by the same stain metachromatically but after the effect of heat or an acidic or alkaline reaction orthochromatically. It is thus possible to distinguish the native from the denatured DNA in a smear preparation. We assume that changes observed in the nuclei of hepatocytes after the action of denaturing agents may be regarded as changes due to the denaturation of DNA.     

Photoelectric effects of deoxyribonucleic acid

Studies have been made of the d.c. semiconductive behavior of fibers and solid gels of DNA. The semiconductive behavior under high vacuum is similar in solid gels of DNA and stretched fibers of DNA measured along and across the fiber axis. Photoelectric effects have been studied, and an action spectrum is reported which agrees with the absorption spectrum of DNA. No photoelectric effects are observed at wavelengths corresponding to the thermal energy gap. The temperature dependence of the photocurrent has been determined, and the effects of air, oxygen, and nitrogen at different pressures on the photocurrent have been measured.     

Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.