Deoxyribonucleic Acid Repair Synthesis Research Articles

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

The erythemal action spectrum and deoxyribonucleic acid repair synthesis in xeroderma pigmentosum.

The reactions to monochromatic radiation of the skin of ten patients with xeroderma pigmentosum were investigated, and eight were abnormal. The abnormalities consisted of papular and vesicular reactions and delay in the development of the minimal erythema dose reaction with wavelengths principally in the 290-320 nm range. Three patients were too young for full action spectra to be obtained but of those patients in whom all wavelengths were tested only one showed a reaction to radiation above 320 nm and this was at 340 nm only. In only one patient was the minimal erythema dose at 300 nm at 24 h lower than normal. In six patients the repair synthesis of deoxyribonucleic acid after ultraviolet radiation was estimated. Reduced levels were seen in five but repair was normal in one patient. The patient with normal repair also had normal reactions to monochromatic radiation. The abnormal reaction of the skin to artificial radiation and the abnormal deoxyribonucleic acid repair synthesis may enable the diagnosis of xeroderma pigmentosum to be made at a very early age. In one of the patients the diagnosis was made at the age of 6 months in a child with photosensitivity but with no other clinical signs of the disease. It is suggested that, by making the diagnosis as early as possible and by protecting the skin from natural sunlight, cutaneous malignancies may be prevented.

The Initiation of DNA Excision-Repair

Publisher Summary The excision-repair of Deoxyribonucleic acid (DNA) is an enzymatically mediated process by which modified bases or fragments of bases are removed from cellular DNA together with adjoining normal nucleotides. This segment of excised DNA is then resynthesized through the action of a DNA polymerase using the opposite strand as a template. The last phosphodiester bond is sealed through the action of DNA ligase. The importance of this process to human well being through the prevention of cancer was emphasized by the demonstration that in contrast to normal cells, cells of individuals with xeroderma pigmentosum (XP)—a hereditary disease—did not remove ultraviolet (UV) radiation-induced pyrimidine dimers from their nuclear DNA. The importance of the DNA repair system is emphasized by the demonstration that alkylating agents caused the onset of DNA repair synthesis when applied to human (HeLa) cells in culture. Subsequently it became evident that the DNA repair system could affect the removal of many types of modified bases in addition to pyrimidine dimmers and that DNA repair constituted a general defense mechanism against a wide variety of potentially mutagenic and/or carcinogenic DNA modifications.

Effect of histone H1 removal on the distribution of ultraviolet-induced deoxyribonucleic acid repair synthesis within chromatin.

Effect of histone H1 removal on the distribution of ultraviolet-induced deoxyribonucleic acid repair synthesis within chromatin.

Distribution within chromatin of deoxyribonucleic acid repair synthesis occurring at different times after ultraviolet radiation.

We have compared the initial distribution and subsequent redistribution within chromatin of nucleotides incorporated during the early ("rapid") phase and the late ("slow") phase of UV-induced DNA repair synthesis. As has been observed for the early repair phase, most or all of the nucleotides incorporated during the late repair phase are initially staphylococcal nuclease and DNase I "sensitive" (i.e., rapidly digested). This initial enhanced sensitivity is accompanied by both an underrepresentation of these nucleotides in the 145--165 base pari (core) DNA produced by staphylococcal nuclease digestion and an absence of these nucleotides in the approximately 20-base repeat pattern produced by DNase I digestion. Furthermore, nucleotides incorporated at late time after damage are involved in nucleosome rearrangements as reported previously for repair synthesis occurring at early times [Smerdon, M. J., & Lieberman, M. W. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4238--4241]. The kinetics of redistribution, however, appear to be more rapid than those observed for early times. Following redistribution the average nucleosome repeat length of DNA containing repair-incorporated nucleotides is the same as that of bulk DNA regardless of the time after damage that repair occurs; also, many of these nucleotides coelectrophorese with the approximately 10-base repeat fragments generated by DNase I. The results yield a new interpretation of our previous studies [Smerdeon, M. J., Tlsty, T. D., & Lieberman, M. W. (1978) Biochemistry 17, 2377--2386] on the distribution of nucleotides incorporated at long times after UV irradiation.

Rapid assay for repair of damage induced by ultraviolet light in bovine lymphocytes

Bovine peripheral blood lymphocytes exhibit a peak of deoxyribonucleic acid (DNA) repair, measured after exposure of cells to ultraviolet light, three to four days after phytohaemagglutinin addition to diluted blood cultures. This peak of DNA repair is induced coincidentally with that of DNA replication. DNA repair is determined in the presence of hydroxyurea which inhibits DNA replication. This assay provides a rapid screening method for deficits in DNA repair synthesis in cattle.

Deoxyribonucleic acid repair synthesis in permeable human fibroblasts exposed to ultraviolet radiation and N-acetoxy-2-(acetylamino)fluorene.

We have studied deoxyribonucleic acid (DNA) repair synthesis in permeable, confluent normal and repair- deficient human fibroblasts using (3H)dCTP, BrdUTP, dATP, and dGTP as substrates. In this system repair synthesis occurred as judged by the following criteria: (1) labeled nucleotides were incorporated into parental DNA (p = 1.73 g/cm3 in alkaline CsCl); (2) incorporation into parental DNA was negligible in the absence of damage and increased 10- 20-fold in normal cells following exposure to ultraviolet ra- diation (UV) or the direct-acting chemical carcinogen N- acetoxy-2-(acetylamino)fluorene; and (3) damage-dependent DNA synthesis was absent in preparations made from excision repair-deficient human diploid fibroblasts (xeroderma pig- mentosum cells, complementation group A). The reaction was

Gap-filling repair synthesis induced by ultraviolet light in a Bacillus subtilis Uvr- mutant.

Deoxyribonucleic acid repair synthesis was studied in one wild-type and two mutant strains of Bacillus subtilis that are defective in excision of pyrimidine dimers. The cells were irradiated with ultraviolet light, and 6-(p-hydroxyphenyl-azo)-uracil was used to block replicative synthesis, allowing only repair synthesis. One of the mutations (uvs-42) resulted in a severe inhibition of incision, dimer excision, and repair synthesis. In contrast, the other mutant (uvr-1) slowly incised and excised dimers and did repair synthesis in patches which appear to be several-fold longer than those in the wild-type strain, apparently because large gaps are produced at excision sites. The results indicate that the primary defect in uvs-42 cells is in initiation of dimer excision, whereas the uvr-1 mutation appears to be a defect in the exonuclease normally used to complete dimer excision.

Damage of proliferating lymphoid cell deoxyribonucleic acid by methyl methanesulfonate and N-acetoxy-2-acetylaminofluorene

Replicative synthesis of deoxyribonucleic acid by splenic lymphoid cells cultured from C57BL/6J strain mice and stimulated to proliferate with concanavalin A (Con A) was strongly suppressed by short exposure (10-60 min) of the cells to the direct-acting carcinogens methyl methanesulfonate or N-acetoxy-2-acetylaminofluorene. Carcinogen-treated lymphoid cells failed to recover stimulated levels of replicative synthesis after incubation for several hours in fresh medium without carcinogen. In contrast, spleen cells treated with hydroxyurea (a metabolic inhibitor of deoxyribonucleic acid replication) fully recovered stimulated rates of replicative synthesis upon incubation in medium without hydroxyurea. Furthermore, the inhibition of replicative synthesis by the carcinogens was accompanied by marked stimulation of deoxyribonucleic acid repair synthesis. Concomitant measurement of replicative and repair synthesis appears to be an effective approach for the in vitro study of carcinogen-induced damage in the deoxyribonucleic acid of proliferating lymphoid cells.

Deoxyribonucleic acid repair synthesis: a new factor for defining high blood pressure.

1. The level of DNA repair synthesis has been compared in males diagnosed as having hypertension (diastolic blood pressure greater than or equal to 100 mmHg), males with slightly elevated blood pressure (greater than 95th percentile corrected for age) and males with normal or subnormal blood pressure (less than or equal to 30th percentile corrected for age). 2. The hypertensive males and the males with elevated blood pressure could not be distinguished from each other with respect to the levels of chemically induced repair synthesis, but both were significantly increased over the induced repair synthesis values of individuals with normal or low blood pressure. Since it has been shown previously that lymphocytes with high repair synthesis values also have increased levels of both carcinogen-DNA binding and chromosomal aberrations (Nordén, Scherstén, Thulin, Pero, Bryngelsson & Mitelman, 1975), the biological significance of this variable appears to be of value in discriminating between normal and high blood pressure groups.

Role for deoxyribonucleic acid ligase in deoxyribonucleic acid polymerase i-dependent repair synthesis in toluene-treated escherichia coli

In a toluene-treated mutant of Escherichia coli K-12 having a temperature-sensitive, conditionally lethal mutation in the structural gene for deoxyribonucleic acid (DNA) ligase, an extensive DNA repair synthesis occurred in X-irradiated cells at the nonpermissive temperature, 42 C. At the permissive temperature, 30 C, nearly normal semiconservative synthesis and limited repair synthesis were observed when DNA ligase was activated by the addition of nicotinamide adenine dinucleotide.

Distribution of deoxyribonucleic acid repair synthesis among repetitive and unique sequences in the human diploid genome.

Distribution of deoxyribonucleic acid repair synthesis among repetitive and unique sequences in the human diploid genome.

Photoreactivation and Excision Repair of Ultraviolet Radiation-Injured DNA in Primary Embryonic Chick Cells

Primary embryonic chick cells have been evaluated on the basis of their capacity to repair photochemical lesions produced in the deoxyribonucleic acid (DNA) by ultraviolet (UV) radiation. The fate of one prominent class of UV photoproducts, cyclobutane pyrimidine dimers, was monitored by an in vitro enzymatic assay. UV-irradiated cultures were incubated for prescribed times after which their damaged, radioactive-labeled DNA was extracted and exposed to a purified UV endonuclease selectively active toward sites altered by dimer formation. Single-strand scissions specifically introduced by the enzyme treatment and, therefore, the dimer-containing sites remaining in the DNA were quantified retrospectively by velocity sedimentation in alkaline sucrose. When the chick fibroblasts were incubated in black light, essentially all nuclease-susceptible sites rapidly disappeared from the UV-damaged DNA. In sharp contrast, incubation of the irradiated cultures in total darkness severely impeded the metabolic machinery responsible for site elimination. A substantial amount of UV-stimulated DNA repair synthesis was also detected in the chick cells by conventional techniques involving isopyknic centrifugation and autoradiography. However, the UV photoproducts triggering this indicator of excision repair were probably not dimers since incubation of the irradiated cultures in the light rather than in the dark did not lead to a diminution in the extent of repair synthesis. By these criteria of DNA repair, it appears that embryonic chick cells primarily rely on a highly proficient, light-requiring mechanism, presumably enzymatic photoreactivation, for dimer elimination but also possess a light-independent, excision-type process to cope with other, as yet unidentified, photochemical defects.