A difference in the pattern of repair in a large genomic region in UV-irradiated normal human and Cockayne syndrome cells.

Xeroderma pigmentosum group C cells repair DNA damaged by ultraviolet radiation in an unusual pattern throughout the genome. They remove cyclobutane pyrimidine dimers only from the DNA of transcriptionally active chromatin regions and only from the strand that contains the transcribed strand. The repair proceeds in a manner that creates damage-free islands which are in some cases much larger than the active gene associated with them. For example, the small transcriptionally active beta-actin gene (3.5 kb) is repaired as part of a 50 kb single-stranded region. The repair responsible for creating these islands requires active transcription, suggesting that the two activities are coupled. A preferential repair pathway in normal human cells promotes repair of actively transcribed DNA strands and is coupled to transcription. It is not known if similar large islands, referred to as repair domains, are preferentially created as a result of the coupling. Data are presented showing that in normal cells, preferential repair in the beta-actin region is associated with the creation of a large, completely repaired region in the partially repaired genome. Repair at other genomic locations which contain inactive genes (insulin, 754) does not create similar large regions as quickly. In contrast, repair in Cockayne syndrome cells, which are defective in the preferential repair pathway but not in genome-overall repair, proceeds in the beta-actin region by a mechanism which does not create preferentially a large repaired region. Thus a correlation between the activity required to preferentially repair active genes and that required to create repaired domains is detected. We propose an involvement of the transcription-repair coupling factor in a coordinated repair pathway for removing DNA damage from entire transcription units.

Definition of a DNA repair domain in the genomic region containing the human p53 gene.

The human p53 gene is repaired in UV (254 nm)-irradiated xeroderma pigmentosum group C (XP-C) cells as part of a large genomic region that is about twice the size of the gene. Surrounding genomic regions are not repaired. Through DNA cloning and measurements of DNA repair, we mapped the location of the repair domain, including the terminal regions, relative to the topological features of the gene. The domain includes only the DNA strand that is transcribed and extends in both 3' and 5' directions beyond the promoter and transcription termination sites. No transcriptional activity other than that associated with the p53 gene was detected. The results suggest that nontranscribed regions adjacent to the p53 transcribed regions are efficiently repaired in XP-C cells. This means that factors associated with transcription other than RNA polymerase II and the associated transcription repair coupling factor must also play a role in the selective repair process in XP-C cells. We also found that a DNA fragment that contains the p53 promoters is nearly twice as sensitive to cyclobutane pyrimidine dimer induction by UV irradiation than are the surrounding fragments, which have the expected sensitivity.

Identification of a large genomic region in UV-irradiated human cells which has fewer cyclobutane pyrimidine dimers than most genomic regions.

Size separation after UV-endonuclease digestion of DNA from UV-irradiated human cells using denaturing conditions fractionates the genome based on cyclobutane pyrimidine dimer content. We have examined the largest molecules available (50-80 kb; about 5% of the DNA) after fractionation and those of average size (5-15 kb) for content of some specific genes. We find that the largest molecules are not a representative sampling of the genome. Three contiguous genes located in a G+C-rich isochore (tyrosine hydroxylase, insulin, insulin-like growth factor II) have concentrations two to three times greater in the largest molecules. This shows that this genomic region has fewer pyrimidine dimers than most other genomic regions. In contrast, the beta-actin genomic region, which has a similar G+C content, has an equal concentration in both fractions as do the p53 and beta-globin genomic regions, which are A+T-rich. These data show that DNA damage in the form of cyclobutane pyrimidine dimers occurs with different probabilities in specific isochores. Part of the reason may be the relative G+C content, but other factors must play a significant role. We also report that the transcriptionally inactive insulin region is repaired at the genome-overall rate in normal cells and is not repaired in xeroderma pigmentosum complementation group C cells.

Repair of some active genes in Cockayne syndrome cells is at the genome overall rate.

Repair of UV (254 nm)-induced DNA damage in cells from patients with the genetic disease Cockayne syndrome (CS; CS3BE, CS2BE) has been examined in several different genomic regions. These regions include those that contain the actively transcribed beta-actin and adenosine deaminase (ADA) genes and the inactive insulin and 754 loci. The beta-actin, ADA and insulin regions are repaired at about the same rate, one which is equal to the genome overall repair rate. The 754 locus is repaired considerably more slowly. The insulin region is repaired at the same rate in both CS and normal cells as is the 754 locus. The only difference from normal is that the active genes, while repaired well, are not preferentially repaired relative to the genome overall. Our results are consistent with the hypothesis that the repair defect in CS is due to an inactive transcription-repair coupling factor (TRCF). However, the results also indicate that factors other than TRCF and active transcription must also promote repair of some regions relative to others in both normal and CS cells.

DNA repair in the genomic region containing the beta-actin gene in xeroderma pigmentosum complementation group C and normal human cells.

The limited DNA excision repair in UV-irradiated fibroblasts from xeroderma pigmentosum complementation group C (XP-C) occurs in selected chromatin regions. The small beta-actin gene (3.5 kb) is one of these and is repaired as part of a large region (about 50 kb). We show here that only one of the DNA strands is repaired through this extended region. Several genomic fragments spanning about 70 kb in the beta-actin region have been cloned and mapped and some have been examined for repair activity. Both strands of one fragment (14 kb) in the immediate vicinity of the gene are repaired. Transcripts associated with both strands are detected. In normal cells, both strands of the same fragment are preferentially repaired relative to the genome overall and also associated with transcription. The repair activity in XP-C cells associated with other defined DNA fragments indicates that termini for the repaired regions in either strand can be located. Results are consistent with those of others indicating that transcription promotes repair in XP-C cells and that several levels of repair activity, at least one coupled to transcription, occur in normal cells. We conclude that the beta-actin repair domain, defined in XP-C cells, comprises both strands of a small region (about 14 kb) in the vicinity of the beta-actin gene and a single strand extending through a larger region of about 50 kb. We suggest that a similar genomic organization for repair exists in normal cells.

A re-examination of the intragenome distribution of repaired sites in proliferating xeroderma pigmentosum complementation group C fibroblasts.

We find that rapidly proliferating fibroblasts from xeroderma pigmentosum complementation group C (XP-C) patients, cells that have a small residual DNA excision repair capacity, repair DNA in localized regions of the genome in a clustered pattern rather than at single sites in dispersed locations. This finding is similar to that observed earlier for nondividing cells but is in contrast to published results that indicate that the residual repair in proliferating XP-C cells is dispersed throughout the genome in a non-clustered pattern. While we detect the same amount of repair in both proliferating and nondividing cells, we also observe no shift from the clustered pattern of repair to a more dispersive pattern when nondividing cells are stimulated to proliferate by fresh serum addition. We have no obvious explanation for these discrepancies with the published results. We have noted previously that proliferating XP-C cells are very UV sensitive relative to normal cells while nondividing cells that exhibit the same amount of repair activity are relatively UV resistant. There is no satisfactory explanation for this change in relative response to the lethal effects of UV, a change not observed for cell strains from other XP complementation groups. However, we argue that clustered repair in specific genomic regions promotes survival in nondividing XP-C cells but does not promote survival in proliferating cells.

Selective repair of specific chromatin domains in UV-irradiated cells from xeroderma pigmentosum complementation group C.

The limited DNA-excision repair in UV-irradiated nondividing fibroblasts from xeroderma pigmentosum complementation group C (XP-C) occurs in localized chromatin regions generating large DNA segments (at least 30-70 kb) free of pyrimidine dimers. A genomic fraction enriched for this DNA was isolated on the basis of the larger size of the repaired fragments after UV-endonuclease treatment and screened for specific genes. It contains more copies per microgram DNA of two transcriptionally active genes, beta-actin and dihydrofolate reductase, compared to the remaining DNA but an equal number of copies per microgram DNA of an inactive locus termed 754. We confirmed that the active genes were preferentially repaired by measuring the removal of pyrimidine dimers from specific genomic restriction fragments comprising these sequences. These results mean that a unique set of relatively large chromatin domains are repaired in nondividing XP-C cells, even though most of the DNA remains unrepaired. The repaired domains may be those containing the active genes. This specific repair may account for the relatively high UV-resistance of the nondividing cells. In normal cells, a very rapid repair of a restriction fragment containing the beta-actin gene and slow repair of the 754-containing fragment was detected indicating that a similar domain-oriented repair process also exists in these cells. These results are consistent with the previously discovered rapid repair of active genes compared to bulk DNA. Separate damage-recognition systems may exist in human cells for chromatin domains that contain transcribed regions and those that contain no transcribed regions. The latter system may be deficient in XP-C.

Biological significance of domain-oriented DNA repair in xeroderma pigmentosum cells.

The patterns (domain oriented versus a random location) and amounts of DNA excision repair, determined by standard density gradient techniques and sedimentation properties of partially repaired and UV-endonuclease-digested DNA in alkaline sucrose gradients, are reported for UV (254 nm)-irradiated nondividing xeroderma pigmentosum complementation group C or A (XP-C, XP-A) and normal cells. Repair synthesis in relatively UV-resistant XP-C (XP4RO) cells is domain oriented and limited (10% of normal values) while it is randomly located and not as limited in more sensitive XP-A (XP8LO) cells. Thus, greater UV resistance is associated with a very limited but domain-oriented pattern of repair. In XP-C cells, both total and domain-oriented repair syntheses, while limited, increase with UV dose and plateau at about 15-20 J/m2, as observed for normal cells. We suggest that repair in XP-C is limited at the lower UV doses (less than 15-20 J/m2) by substrate levels in specific chromatin domains and not by availability of essential enzymes for domain-oriented repair. In contrast, the XP-A strain XP8LO exhibits normal repair activities for doses up to 5 J/m2 and limited repair at higher doses, indicating that repair occurs through normal pathways that are limited by reduced availability of an essential enzyme.

The endogenous nuclease sensitivity of repaired DNA in human fibroblasts.

The limited DNA excision repair that occurs in the chromatin of UV-irradiated growth arrested cells isolated from a xeroderma pigmentosum (XP) complementation group C patient is clustered in localized regions. The repaired DNA was found to be more sensitive to nicking by endogenous nucleases than the bulk of the DNA. The extra-sensitivity does not change with increasing amounts of DNA damage or repair activity in the locally-repaired regions and is retained through a 24-h chase period. We suggest that these results are due to the occurrence of DNA repair limited to pre-existing, non-transient chromatin fractions that contain actively transcribed DNA. A similar extra-sensitivity of repaired DNA was not detected in cells of normal or XP complementation group A strains that exhibit either normal or limited repair located randomly throughout their genomes. The association between endogenous nuclease sensitivity and clustered repair probably defines a normal excision repair pathway that is specific for selected chromatin domains. The repair defect in XP-C strains may be one in pathways targeted for other endogenous nuclease-resistant domains.

A further definition of characteristics of DNA-excision repair in xeroderma pigmentosum complementation group A strains.

The location in the genome of excision repair following exposure to UV (254 nm) of two XP complementation group A strains, XP12BE and XP8LO, that differ considerably in their excision-repair rates, have been determined. Capacity for repair in XP8LO has also been determined. Sites repaired in DNA in a 24-h post-UV period were located relative to the remaining pyrimidine dimers using the M. luteus UV-endonuclease to nick partially repaired DNA and sedimentation in alkaline sucrose to size the resulting DNA. Repair in group A occurs randomly throughout the genome in a manner similar to that observed for normal cells but in contrast to domain-limited repair in group C strains. This observation defines a further similarity of the excision repair detected in group A compared to normal cells that is in addition to the previously reported related characteristics of the respective excision rate curves. A reduced repair capacity in XP8LO relative to normal cells was detected. This strain, which repairs DNA at an initial rate identical to that of normal strains when irradiated with doses of 5 J/m2 or less, repairs DNA at a slower than normal but constant rate at higher doses. This leads to the suggestion that XP8LO is defective in the number of repair enzyme complexes compared to normal cells.

The rate of removal of pyrimidine dimers in quiescent cultures of normal human and xeroderma pigmentosum cells.

The rate of removal of pyrimidine dimers from DNA of UV (254 nm)-irradiated (1 J/m2) normal and xeroderma pigmentosum (XP) cells maintained in culture as nondividing populations was determined. Several normal and XP strains from complementation groups A, C and D were studied. The excision rates and survival ability of nondividing cells were examined to determine if an abnormal sensitivity was associated with a decreased rate of dimer excision. The results show that all normal strains studied excise pyrimidine dimers at the same rate, with the rate curve characterized by two components. All 'excision-deficient' XP strains excise dimers at a slower-than-normal rate, with the rate curves also characterized by two components. The rate constants for the first components of all of the XP strains (group A, C and D) are the same, one tenth of the normal rate constant, except for XP8LO (group A). XP8LO has a first-component rate constant similar to that of normal strains and a second component rate constant similar to that of other group A strains (XP12BE, XP25RO). Thus, the slower rate of dimer excision in XP8LO is due to a defect in the mechanism responsible for the second component of the excision-rate curve. In general, an abnormal sensitivity of nondividing cells to UV is associated with a reduced dimer-excision rate. A notable exception to this is the group C strain XP1BE which has an initial repair rate similar to that of group A XP12BE but is considerably more resistant when survival is measured.

Evidence that DNA excision-repair in xeroderma pigmentosum group A is limited but biologically significant.

The loss of pyrimidine dimers in nondividing populations of an excision-repair deficient xeroderma pigmentosum group A strain (XP12BE) was measured throughout long periods (up to 5 months) following exposure to low doses of ultraviolet light (UV, 254 nm) using a UV endonuclease-alkaline sedimentation assay. Excision of about 90% of the dimers induced by 1 J/m2 occurred during the first 50 days. The rate curve has some similarities with that of normal excision-repair proficient cultures that may not be coincidental. Rate curves for both XP12BE and normal cultures are characterized by a fast and slow component, with both rate constants for the XP12BE cultures (0.15 day-1 and 0.025 day-1) a factor of 10 smaller than those observed for the respective components of normal cell cultures. The slow components for both XP12BE and normal cultures extrapolate to about 30% of the initial number of dimers. No further excision was detected throughout an additional 90-day period even though the cultures were capable of excision-repair of other newly-introduced pyrimidine dimers. We conclude that nondividing XP12BE cells in addition to having a slower repair rate, cannot repair some of the UV-induced DNA damage. The repair in XP12BE is shown to have biological significance as detected by a cell-survival assay and dose-fractionation techniques. Nondividing XP12BE cells are more resistant to UV when irradiated chronically than when irradiated acutely with the same total dose.

Rate and extent of DNA repair in nondividing human diploid fibroblasts.

Rates of DNA repair in ultraviolet (254 nm)-irradiated nondividing human diploid fibroblasts were determined at doses as low as 1 J/sq m using an enzymatic assay for pyrimidine dimers. In normal cells, initial rates (dimers removed per 24 hr) increased with dose to 20 J/sq m with no further increase at 40 J/sq m. At 10 J/sq m or less, repair occurred continuously over long postultraviolet periods until all the damage that could be detected was removed (for 10 J/sq m, this required 20 days; sensitivity of the assay was about 0.1 dimer/10(8) daltons). The overall rate curves appear as the sum of two first-order reactions with different rate constants (rapid, 1.7 dimers/10(8) daltons/day; slow, 0.25 dimer/10(8) daltons/day). The slow reaction extrapolates to 30 to 40% of the original dimers. Populations irradiated a second time after greater than 90% of the original damage had been removed repaired the newly added DNA damage with similar kinetics and to the same extent. Repair kinetics in a xeroderma pigmentosum strain (XP12BE, Complementation Group A, 1 J/sq m) lacks the rapid component and approximates the slow component of normal cells. If the slow component of normal cells is due to repair of less accessible dimers, as suggested by others, then by analogy, slow excision repair in XP12BE may be due to the poor accessibility of all dimers. This suggests that the XP12BE excision repair defect is in the enzymes that render dimers in chromatin accessible to repair.