Bernard S. Strauss

Sites of inhibition of in vitro DNA synthesis in carcinogen- and UV-treated phi X174 DNA.

THE availability of DNA molecules of known sequence and rapid methods for the determination of the sequence of particular DNA fragments1–3 make it possible to investigate the molecular action of mutagens and carcinogens more closely4,5. For example, there is considerable evidence that some types of lesion in DNA such as UV light-induced pyrimidine dimers and various chemical adducts are blocks to DNA synthesis both in vivo6–9 and in vitro10,11. The available data support the hypothesis that the block occurs at the level of the lesion on the template strand10,11. We have used DNA containing such lesions on a template of known sequence for in vitro DNA synthesis by DNA polymerase 1 to investigate the site of the block at the level of the nucleotide sequence. This method allows us to determine both the site at which lesions capable of blocking synthesis by DNA polymerase occur and exactly where synthesis stops in relation to the lesion.

The role of DNA polymerase in base substitution mutagenesis on non-instructional templates

In vitro DNA synthesis on phi X174 or M13 templates with non-instructional lesions such as UV dimers or AP (apurinic/apyrimidinic) sites terminates one base before the site of the lesion when synthesis is catalyzed by T4 DNA polymerase or E. coli polymerase I. E. Coli polymerase I also produces termination bands at the site of AP lesions. Substitution of Mn2+ for Mg2+ and increasing the concentration of dNTPs results in elongation of the newly synthesized strand opposite the site of the lesion and beyond. Purine deoxynucleoside triphosphates are utilized for insertion opposite lesions to a greater extent than are pyrimidine deoxynucleoside triphosphates. Deoxy ATP is used almost exclusively for elongation opposite AP sites with pol I-Klenow fragment in the presence of Mg2+. We suppose that these results illustrate the previously observed greater affinity of polymerases under template-free conditions for purine nucleotides. We also suppose that the results can be used to account for mutagenic base selection on noninstructional DNA templates. If purines are preferentially selected by polymerases, then treatments which inactivate pyrimidines will lead to an excess of transitions whereas inactivation of purines will produce more transversions. Data in the literature support this hypothesis.

Accumulation of single-stranded regions in DNA and the block to replication in a human cell line alkylated with methyl methane sulfonate

DNA synthesized by cells from a human carcinoma of the larynx (HEp.2 cells) contains regions of single strandedness as judged by chromatography with benzoylated naphthoylated DEAE-cellulose and sensitivity to single-strand specific nucleases. DNAss† is observed after 10 or 30-minute labeling periods and the label becomes associated with native DNA after further incubation. Hydroxyurea induces an increase in the proportion of DNAss and this increase is correlated with the inhibition of DNA synthesis. There is a dose-dependent increase in the proportion of DNAss synthesized by HEp.2 cells after alkylation with MMS. Addition of hydroxyurea prevents the conversion of DNAss into native DNA in both control and alkylated cells. Single-stranded regions are found in parental DNA and the total extent of single strandedness is greater in parental than in daughter DNA. The amount, as opposed to the proportion, of single strandedness is only slightly greater in alkylated than in control cultures. DNA synthesized eight hours after treatment with MMS has the same proportion of DNAss as the control. Repair synthesis can be measured as the MMS-induced incorporation of thymidine in the presence of hydroxyurea. An MMS-induced increase in thymidine incorporation in the caffeine eluate from BND-cellulose indicates repair synthesis in DNAss. We interpret these data as follows: (a) the single-stranded regions are normal intermediates, which occur transiently at the growing point as a result of discontinuous DNA synthesis; (b) fragments of daughter DNA become single stranded as a result of strand displacement; (c) the growing point is held up by the alkylation lesion; (d) repair synthesis in the growing point region removes the alkylation lesion and permits DNA synthesis to continue.

Repair of damage induced by a monofunctional alkylating agent in a transformable, ultraviolet-sensitive strain of Bacillus subtilis.

An ultraviolet-sensitive, transformable strain of Bacillus subtilis (uvr−) was unable to carry out host cell reactivation of the virulent bacteriophage SP01. The ratio of the slopes (kuvr-/kuvr+) of the ultraviolet-inactivation curves was 6 to 8 for cells or for whole phage, and 2 to 3 for indole+ transforming DNA or for SPOl phage DNA measured in a transforming system. Whole phage inactivated with nitrogen mustard showed greater survival on uvr+ cells than on uvr− ; there was no such difference for whole phage inactivated with methyl methanesulfonate. Whole uvr+ cells recovered from ultraviolet-induced damage, as determined by an increase in extractable transforming activity during a period of incubation in which net DNA synthesis did not occur. Uvr− cells were unable to recover from ultraviolet-induced damage but they did recover from methyl methanesulfonate-induced damage in similar experiments. Density-labeled [2H15N] methyl methanesulfonate-treated uvr+ cells incubated in [1H14N]medium containing [3H]thymidine showed a threefold increase in extractable transforming activity, with no formation of new DNA molecules as determined by pyenometric analysis. The repair of damage induced by ultraviolet irradiation differs by at least one step from the repair of damage induced by methyl methanesulphonate.

The measurement of chemically-induced DNA repair synthesis in human cells by BND-Cellulose chromatography ☆

Repair synthesis in human cells in tissue culture can be readily separated from semi-conservative DNA synthesis with the aid of a benzoylated naphthoylated DEAE cellulose (BND-cellulose) column. Cells are incubated with a radioactive DNA precursor during treatment with a repair-inducing agent. An inhibitor of semi-conservative DNA synthesis (hydroxyurea) is added to slow the progression of the DNA growing point. The cells are lysed and after treatment with ribonuclease and pronase the lysates are sheared and passed through a BND-cellulose column. Native DNA is eluted with I M NaCl. Any increase in radioactivity in the native DNA is due to repair synthesis and the specific repair activity (nucleotides inserted per mug of DNA) can be determined from radioactivity and absorbancy measurements. Repair can also be measured in the region of the DNA growing point by fractionation of the material eluted from BND-cellulose with 50% formamide. Repair was not detected in N-acetoxy-2-acetylaminofluorene (AAAF)-treated lymphoblasts derived from an individual with xeroderma pigmentosum although methyl methanesulfonate (MMS)-induced repair was observed in these cells.

DNA methylated in vitro by a monofunctional alkylating agent as a substrate for a specific nuclease from Micrococcus lysodeikticus

Abstract Coliphage T7 DNA was alkylated with methyl methanesulfonate to give an alkali-stable product with about 16 added methyl groups per molecule. This methylated DNA was attacked by an extract from Micrococcus lysodeikticus which made single strand breaks in methylated but not in control DNA. The breaks occurred at or near the site of the methylation. Bacillus subtilis extracts also attacked methylated DNA but in contrast to the M. lysodeikticus preparations were unable to attack ultraviolet-treated DNA, suggesting that separate enzymes recognize the different types of damage. Methylation per se converts DNA into a substrate for a specific nuclease.

The intermediate in the degradation of DNA alkylated with a monofunctional alkylating agent

Abstract The course of DNA degradation following alkylation with a monofunctional alkylating agent can be accounted for by the scheme: A → k 1 B → k 2 where A represents alkylated DNA, B represents DNA with apurinic sites, and C indicates DNA containing single-strand breaks. Apurinic sites in DNA were estimated as the fraction of the DNA made acid soluble by treatment with alkali. A simple method for comparing the rate of depurination for DNA alkylated with different groups is described. The rate of hydrolysis of apurinic sites depended on the buffer; buffers with primary amino groups such as Tris or glycinamide promoted hydrolysis of apurinic acid at a pH near neutrality. Alkaline phosphatase-sensitive groups were exposed as a result of the degradation of DNA. The proportion of phosphatase-sensitive to acid-soluble groups remained constant throughout the course of the degradation of methylated or ethylated DNA. We conclude that apurinic acids are necessary intermediates in the degradation in vitro of alkylated DNA. Ethylated and methylated DNAs decay by the same reaction mechanism.

The presence of breaks in the deoxyribonucleic acid of Bacillus subtilis treated in vivo with the alkylating agent, methylmethanesulfonate

Abstract DNA was alkylated with methylmethanesulfonate in vitro, or in vivo by treating suspensions of Bacillus subtilis with methylmethanesulfonate. Both reversible and irreversible thermal denaturation curves were determined as well as specific transforming activity. Comparison of the results with similar determinations on transforming DNA from B. subtilis treated with pancreatic DNAase indicated that the loss of transforming activity in DNA extracted from alkylated organisms could be accounted for by the insertion of single strand breaks into the DNA. Evidence was presented that these breaks were inserted in vivo by a nuclease which acts on alkylated but not normal DNA. DNA alkylated in vitro with methylmethanesulfonate can be inactivated for transformation by heating at 50° and there is some evidence that this inactivation occurs by depurination rather than by the insertion of single strand breaks.

Frameshift mutation, microsatellites and mismatch repair

The structure of eukaryotic DNA, with its repeated sequences, makes base addition and loss a major obstacle to the maintenance of genetic stability. As compared to the bacteria, much of the mismatch repair capacity of the eukaryotic cell must be devoted to the surveillance of frameshift changes. Any alteration in the activity of proteins which recognize frameshifts or which hold the DNA in place during replication is likely to result in genomic instability.The structure of eukaryotic DNA, with its repeated sequences, makes base addition and loss a major obstacle to the maintenance of genetic stability. As compared to the bacteria, much of the mismatch repair capacity of the eukaryotic cell must be devoted to the surveillance of frameshift changes. Any alteration in the activity of proteins which recognize frameshifts or which hold the DNA in place during replication is likely to result in genomic instability.

Cellular Aspects of Dna Repair

DNA repair reactions are under cellular control. In bacteria, the reactions removing 0(6)-methylguanine and 3-methyladenine are inducible. It is not clear whether similar inducibility occurs in human lymphoblastoid cells. Nonetheless, the ability to manufacture the 0(6)-methylguanine acceptor protein does seem to be controlled by some chromosomal mechanism which is superimposed on the structural gene. This control system may affect reactions other than the removal of 0(6)-methylguanine. Insofar as this is so, transformed human lymphoblastoid cells have a system reminiscent of that found in bacteria.