Evidence for inactivation of DNA repair in frozen and thawed mammalian cells.

A variety of cell strains and lines were frozen and thawed by conventional techniques for cell storage. Following thawing, extracts of cells were prepared and incubated with UV-irradiated E. coli DNA. Thymine dimer excision activity present in extracts of unfrozen cells was lost in extracts of recently thawed cells. The ability to exercise dimers was restored after about 40 h post-thawing, but the recovery was inhibited if cells were cultured in the presence of puromycin. Correlating with the loss of dimer excising activity there was a reduced cell viability as measured by trypan blue dye exclusion.     

Evaluation of morpholine, 3-morpholinone, and N-substituted morpholines in the rat hepatocyte primary culture/DNA repair test

Although mature mammalian sperm are incapable of DNA repair, repair of damaged sperm DNA can occur after fertilization, as the sperm head decondenses and forms the male pronucleus. To quantify the cytogenetic effects of damage to sperm DNA we adapted the sister-chromatid exchange (SCE) test for use in early mouse embryos. After ultraviolet (UV) irradiation of sperm, eggs were fertilized in vitro and cultured for 2 cell cycles in medium containing fluorodeoxyuridine and bromodeoxyuridine; chromosomes were then prepared for SCE analysis. We found that UV-induced SCEs could be detected at the second cleavage division, and that eggs of different strains showed different frequencies of SCEs when fertilized by damaged sperm of a single strain. These results may indicate strain-specific differences in DNA repair of UV-induced DNA lesions by the early mouse embryo.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

Small circular DNA complexes in eucaryotic cells

A small number of eucaryotic cells (100 to 1000 cells) were pressed by mica sheet; then the extruded contents were adsorbed on mica and processed for electron microscopy. In the absence of divalent cation, small polydisperse circular DNA molecules bound to proteins or membrane material were preferentially adsorbed. The small circular DNA complexes have been found in every eucaryotic cell, primary lymphoid tissue cells of bursa and thymus, primary cell lines of retina and liver, and established cultured cell lines of embryonal teratocarcinoma, F9 and PCC3, HeLa and 3T6. Size distribution of these DNA complexes varies, depending on the cell source. The circles less than 1 μm in contour length predominate in cultured cell lines and the larger ones in primary cell lines and cells in situ. Polydisperse covalently closed circular DNAs were recovered from thymus lymphocytes by the conventional dye-CsCl buoyant density method. Their size distribution was similar to that of the small circular DNA complexes detected by the mica-press-adsorption method. They are present in several tens to hundreds of copies per cell representing, at a maximum, 0.02% of the total cellular DNA. The possibility that small circular DNA complexes may result from gene rearrangement as well as from replicon “misfiring” (A. Varshavsky, 1981, Proc. Nat. Acad. Sci. USA 78, 3673–3677) are discussed.     

Detection of DNA methylation in eucaryotic cells

The methods of molecular biology allow for analyzing the methylation pattern in the whole genome and in particular genes. We differentiate methylated sequences from unmethylated ones by means of cutting the genomic template with methylation-sensitive restriction enzymes or by sodium bisulfite DNA modification. Chemical modification precedes most quantitative and qualitative PCR techniques: MS-PCR, MS-nested PCR, Real-Time PCR, QAMA, HeavyMethyl, MSHRM. Restriction enzymes, on the other hand, may be used together with PCR or hybridisation methods (Southern blot and microarrays). PCRs are conducted with primers specific for methylated and unmethylated sequences and sometimes, similarly to hybridisation techniques, with specifically labeled probes or dyes intercalating to double-stranded nucleic acids. The most advanced methylation detection techniques (MALDI-TOF MS and HPLC) significantly reduce the amount of biological material used for tests, but they require specialist equipment.     

recA-dependent DNA repair processes

UV-radiation-induced lesions in DNA result in the formation of: (1) excision gaps (i.e. a lesion is excised, leaving a gap), (2) daughter-strand gaps (i.e. a lesion can be skipped during replication, leaving a gap), and (3) double-strand breaks (i.e. the DNA strand opposite a gap can be cut). In Escherichia coli, the recA gene product is involved in repairs of all three types of lesions--repair of daughter-strand gaps (2) and double-strand breaks (3) constitutes post-replication repair. The evidence suggests, furthermore, that recA-dependent repair of excision gaps (1) produced in DNA replicated prior to UV irradiation (pre-replication repair) appears to occur by similar mechanisms.     

A fixed site of DNA replication in eucaryotic cells

We studied the role of the nuclear matrix (the skeletal framework of the nucleus) in DNA replication both in vivo and in a cell culture system. When regenerating rat liver or exponentially growing 3T3 fibroblasts are pulse-labeled with ³H-thymidine and nuclear matrix is subsequently isolated, the fraction of DNA remaining tightly attached to the matrix is highly enriched in newly synthesized DNA. After a 30 sec pulse labeling period and limited DNAase I digestion, the matrix DNA of 3T3 fibroblasts, which constitutes 15% of the total DNA, contains approximately 90% of the labeled newly synthesized DNA. Over 80% of this label can be chased out of the matrix DNA if the pulse is followed by a 45 min incubation with excess unlabeled thymidine. These and other kinetic studies suggest that the growing point of DNA replication is attached to the nuclear matrix. Studies measuring the size distribution of the matrix DNA also support this conclusion. Reconstitution controls and autoradiographic studies indicate that these results are not due to preferential, nonspecific binding of nascent DNA to the matrix during the extraction procedures. Electron microscopic autoradiography shows that, as with intact nuclei, sites of DNA replication are distributed throughout the nuclear matrix. A fixed site of DNA synthesis is proposed in which DNA replication complexes are anchored to the nuclear matrix and the DNA is reeled through these complexes as it is replicated.     

Age-related changes in unscheduled DNA synthesis by rat hepatocytes

Ultraviolet-induced unscheduled DNA synthesis was studied as a function of age in hepatocytes isolated from 6- to 32-months-old rats. Unscheduled DNA synthesis was measured by both DNA specific activity and autoradiography. Using both procedures, a significant decline in unscheduled DNA synthesis was observed after 14 months of age.     

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.     

Increased repair in DNA growing point regions after treatment of human lymphoma cells with N-methyl-N'-nitro-N-nitrosoguanidine

Benzoylated naphthoylated DEAE-cellulose columns can be used to separate DNA growing point regions from the bulk of the DNA. We used the columns to estimate DNA excision repair in both fractions. Repair induced by acetoxy acetyl aminofluorene (AAAF), bromomethyl benz(alpha) anthracene (BMBA), and methyl methanesulfonate (MMS) occurs to an equal extent in growing point and non-replicating regions of the DNA. Excision repair induced by methyl nitrosourea (MNNU) and methyl nitronitrosoguanidine (MNNG) occurs to a greater extent in growing point regions of the DNA. The overall amount of methyl nitronitrosoguanidine-induced alkylation is the same for replicating and non-replicating regions of the DNA treated in vitro. We conclude that there is some special interaction between methyl-nitronitrosoguanidine and the growing point region in vivo. We suppose that strand displacement and branch migration return DNA lesions at the growing point to a double stranded configuration at which repair is possible.     

DNA excision in repair proficient and deficient human cells treated with a combination of ultraviolet radiation and acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide

Excision repair was measured in normal human and xeroderma pigmentosum group C fibroblasts treated with ultraviolet radiation and the carcinogens acridine mustard (ICR-170) or 4-nitroquinoline 1-oxide (4NQO) by the techniques of unscheduled synthesis, photolysis of bromodeoxyuridine incorporated into parental DNA during repair, and assays of sites sensitive to ultraviolet (UV)-endonuclease. Doses of ICR-170 and 4NQO, low enough not to inhibit unscheduled DNA synthesis (UDS), caused damage to DNA that was repaired by a long patch type mechanism and the rates of UDS decreased rapidly in the first 12 h after treatment. Repair after a combined action of UV plus ICR-170 or UV plus 4NQO was additive in normal cells and no inhibition of loss of endonuclease sensitive sites was detected. In xeroderma pigmentosum (XP) C cells there was less repair after UV plus ICR-170 than after each treatment separately; whereas there was an additive effect after UV plus 4NQO and no inhibition of loss of endonuclease sensitive sites. The results indicate that in normal human fibroblasts there are different rate limiting steps for removal of chemical and physical damages from DNA and that XP cells have a different repair system for ICR-170, not just a lower level, than normal cells. Possibly the same long patch repair system works on 4NQO damage in both normal and XP cells.