Ultraviolet Lesions Research Articles

Exo1 Competes with Repair Synthesis, Converts NER Intermediates to Long ssDNA Gaps, and Promotes Checkpoint Activation

Ultraviolet (UV) light induces DNA-damage checkpoints and mutagenesis, which are involved in cancer protection and tumorigenesis, respectively. How cells identify DNA lesions and convert them to checkpoint-activating structures is a major question. We show that during repair of UV lesions in noncycling cells, Exo1-mediated processing of nucleotide excision repair (NER) intermediates competes with repair DNA synthesis. Impediments of the refilling reaction allow Exo1 to generate extended ssDNA gaps, detectable by electron microscopy, which drive Mec1 kinase activation and will be refilled by long-patch repair synthesis, as shown by DNA combing. We provide evidence that this mechanism maybe stimulated by closely opposing UV lesions, represents a strategy to redirect problematic repair intermediates to alternative repair pathways, and may also be extended to physically different DNA damages. Our work has significant implications for understanding the coordination between repair of DNA lesions and checkpoint pathways to preserve genome stability.

The chromatin remodeling factor BRG1 stimulates nucleotide excision repair by facilitating recruitment of XPC to sites of DNA damage

BRG1 is a catalytic subunit of the human SWI/SNF-like BAF chromatin remodeling complexes. Recent findings have shown that inactivation of BRG1 sensitizes mammalian cells to various DNA damaging agents, including ultraviolet (UV) and ionizing radiation. However, it is unclear whether BRG1 facilitates nucleotide excision repair (NER). Here we show that re-expression of BRG1 in cells lacking endogenous BRG1 expression stimulates nucleotide excision repair of UV induced DNA damage. Using a micropore UV radiation technique, we demonstrate that recruitment of the DNA damage recognition protein XPC to sites of UV lesions is significantly disrupted when BRG1 is depleted. Chromatin immunoprecipitation of the endogenous DDB2 protein, which is involved in recruiting XPC to UV-induced CPDs (cyclobutane pyrimidine dimers), shows that elevated levels of BRG1 are associated with DDB2 in chromatin in response to UV radiation. Additionally, we detected slow BRG1 accumulation at sites of UV lesions. Our findings suggest that the chromatin remodeling factor BRG1 is recruited to sites of UV lesions to facilitate NER in human chromatin.

Nucleotide excision repair–induced H2A ubiquitination is dependent on MDC1 and RNF8 and reveals a universal DNA damage response

Chromatin modifications are an important component of the of DNA damage response (DDR) network that safeguard genomic integrity. Recently, we demonstrated nucleotide excision repair (NER)-dependent histone H2A ubiquitination at sites of ultraviolet (UV)-induced DNA damage. In this study, we show a sustained H2A ubiquitination at damaged DNA, which requires dynamic ubiquitination by Ubc13 and RNF8. Depletion of these enzymes causes UV hypersensitivity without affecting NER, which is indicative of a function for Ubc13 and RNF8 in the downstream UV-DDR. RNF8 is targeted to damaged DNA through an interaction with the double-strand break (DSB)-DDR scaffold protein MDC1, establishing a novel function for MDC1. RNF8 is recruited to sites of UV damage in a cell cycle-independent fashion that requires NER-generated, single-stranded repair intermediates and ataxia telangiectasia-mutated and Rad3-related protein. Our results reveal a conserved pathway of DNA damage-induced H2A ubiquitination for both DSBs and UV lesions, including the recruitment of 53BP1 and Brca1. Although both lesions are processed by independent repair pathways and trigger signaling responses by distinct kinases, they eventually generate the same epigenetic mark, possibly functioning in DNA damage signal amplification.

Structural Basis of UV DNA-Damage Recognition by the DDB1–DDB2 Complex

Ultraviolet (UV) light-induced pyrimidine photodimers are repaired by the nucleotide excision repair pathway. Photolesions have biophysical parameters closely resembling undamaged DNA, impeding discovery through damage surveillance proteins. The DDB1-DDB2 complex serves in the initial detection of UV lesions in vivo. Here we present the structures of the DDB1-DDB2 complex alone and bound to DNA containing either a 6-4 pyrimidine-pyrimidone photodimer (6-4PP) lesion or an abasic site. The structure shows that the lesion is held exclusively by the WD40 domain of DDB2. A DDB2 hairpin inserts into the minor groove, extrudes the photodimer into a binding pocket, and kinks the duplex by approximately 40 degrees. The tightly localized probing of the photolesions, combined with proofreading in the photodimer pocket, enables DDB2 to detect lesions refractory to detection by other damage surveillance proteins. The structure provides insights into damage recognition in chromatin and suggests a mechanism by which the DDB1-associated CUL4 ubiquitin ligase targets proteins surrounding the site of damage.

Cells From Long-Lived Mutant Mice Exhibit Enhanced Repair of Ultraviolet Lesions

Fibroblasts isolated from long-lived hypopituitary dwarf mice are resistant to many cell stresses, including ultraviolet (UV) light and methyl methane sulfonate (MMS), which induce cell death by producing DNA damage. Here we report that cells from Snell dwarf mice recover more rapidly than controls from the inhibition of RNA synthesis induced by UV damage. Recovery of messenger RNA (mRNA) synthesis in particular is more rapid in dwarf cells, suggesting enhanced repair of the actively transcribing genes in dwarf-derived cells. At early time points, there was no difference in the repair of cyclobutane pyrimidine dimers (CPD) or 6-4 photoproducts (6-4PP) in the whole genome, nor was there any significant difference in the repair of UV lesions in specific genes. However, at later time points we found that more lesions had been removed from the genome of dwarf-derived cells. We have also found that cells from dwarf mice express higher levels of the nucleotide excision repair proteins XPC and CSA, suggesting a causal link to enhanced DNA repair. Overall, these data suggest a mechanism for the UV resistance of Snell dwarf-derived fibroblasts that could contribute to the delay of aging and neoplasia in these mice.

Detecting Ultraviolet Damage in Single DNA Molecules by Atomic Force Microscopy

We report detection and quantification of ultraviolet (UV) damage in DNA at a single molecule level by atomic force microscopy (AFM). By combining the supercoiled plasmid relaxation assay with AFM imaging, we find that high doses of medium wave ultraviolet (UVB) and short wave ultraviolet (UVC) light not only produce cyclobutane pyrimidine dimers (CPDs) as reported but also cause significant DNA degradation. Specifically, 12.5 kJ/m 2 of UVC and 165 kJ/m 2 of UVB directly relax 95% and 78% of pUC18 supercoiled plasmids, respectively. We also use a novel combination of the supercoiled plasmid assay with T4 Endonuclease V treatment of irradiated plasmids and AFM imaging of their relaxation to detect damage caused by low UVB doses, which on average produced ∼0.5 CPD per single plasmid. We find that at very low UVB doses, the relationship between the number of CPDs and UVB dose is almost linear, with 4.4 CPDs produced per Mbp per J/m 2 of UVB radiation. We verified these AFM results by agarose gel electrophoresis separation of UV-irradiated and T4 Endonuclease V treated plasmids. Our AFM and gel electrophoresis results are consistent with the previous result obtained using other traditional DNA damage detection methods. We also show that damage detection assay sensitivity increases with plasmid size. In addition, we used photolyase to mark the sites of UV lesions in supercoiled plasmids for detection and quantification by AFM, and these results were found to be consistent with the results obtained by the plasmid relaxation assay. Our results suggest that AFM can supplement traditional methods for high resolution measurements of UV damage to DNA.

Opposing effects of the UV lesion repair protein XPA and UV bypass polymerase η on ATR checkpoint signaling

An essential component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-activating structure is single-stranded DNA. It has been suggested that nucleotide excision repair (NER) can lead to activation of ATR by generating such a signal, and in yeast, DNA damage processing through the NER pathway is necessary for checkpoint activation during G1. We show here that ultraviolet (UV) radiation-induced ATR signaling is compromised in XPA-deficient human cells during S phase, as shown by defects in ATRIP (ATR-interacting protein) translocation to sites of UV damage, UV-induced phosphorylation of Chk1 and UV-induced replication protein A phosphorylation and chromatin binding. However, ATR signaling was not compromised in XPC-, CSB-, XPF- and XPG-deficient cells. These results indicate that damage processing is not necessary for ATR-mediated S-phase checkpoint activation and that the lesion recognition function of XPA may be sufficient. In contrast, XP-V cells deficient in the UV bypass polymerase eta exhibited enhanced ATR signaling. Taken together, these results suggest that lesion bypass and not lesion repair may raise the level of UV damage that can be tolerated before checkpoint activation, and that XPA plays a critical role in this activation.

UV-induced de novo protein synthesis enhances nucleotide excision repair efficiency in a transcription-dependent manner in S. cerevisiae

DNA damage results in the up-regulation of several genes involved in different cellular physiological processes, such as the nucleotide excision repair (NER) mechanism that copes with a broad range of DNA alterations, including the carcinogenic ultraviolet (UV) light-induced pyrimidine dimers (PDs). There are two NER sub-pathways: transcription coupled repair (TCR) that is specific for the transcribed strands (TS) of active genes and global genomic repair (GGR) that repairs non-transcribed DNA sequences (NTD) and the non-transcribed strands (NTS) of expressed genes. To elucidate the role of UV-dependent de novo protein synthesis in nucleotide excision repair in the budding yeast, we investigated the effect of the protein synthesis inhibitor, cycloheximide, on the removal of PDs. Log phase as well as G 1-synchronized cells were treated with the drug shortly before UV irradiation and immediately thereafter, and the repair of damaged DNA was assessed with the high resolution primer extension technique. The results show that in both cellular conditions, the inhibition of UV-dependent de novo protein synthesis by cycloheximide impairs the excision repair of the transcriptionally active GAL10 and URA3 genes, with a greater effect on the non-transcribed strands. This indicates that UV-mediated de novo protein synthesis is required for efficient nucleotide excision repair, but not for the preferential repair of the TSs. On the other hand, cycloheximide did not affect the repair of either strand of the repressed GAL10 gene or the non-transcribed promoter region of the URA3 gene, showing that UV-induced de novo protein synthesis is not required for PD removal from transcriptionally inactive DNA sequences. Together, these data show that despite the fact that NTD and NTSs are normally repaired by the GGR sub-pathway, their requirement for UV-dependent de novo protein synthesis is different, which may suggest a difference in the processing of UV lesions in these non-transcribed sequences of the genome.

HRAD30 mutations in the variant form of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is an autosomal recessive disease characterized by a high incidence of skin cancers. Yeast RAD30 encodes a DNA polymerase involved in the error-free bypass of ultraviolet (UV) damage. Here it is shown that XP variant (XP-V) cell lines harbor nonsense or frameshift mutations in hRAD30, the human counterpart of yeast RAD30. Of the eight mutations identified, seven would result in a severely truncated hRad30 protein. These results indicate that defects in hRAD30 cause XP-V, and they suggest that error-free replication of UV lesions by hRad30 plays an important role in minimizing the incidence of sunlight-induced skin cancers.

TATA-binding protein promotes the selective formation of UV-induced (6-4)-photoproducts and modulates DNA repair in the TATA box.

DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

Persistence and decay of thermoinducible error-prone repair activity in nonfilamentous derivatives of tif-1, Escherichia coli B/r: the timing of some critical events in ultraviolet mutagenesis.

Ultraviolet (UV) mutagenesis in E. coli is associated with a UV-inducible type of error-prone postreplication repair ("SOS" repair) which, in tif-1 strains, is thermo-inducible in coordination with other recA+ lexA+-dependent inducible functions, including filamentous growth. Mutants of E. coli B/r tif-1 strains have been isolated which retain thermoinducibility of SOS repair activity, but lack the thermosensitivity caused by filamentous growth at 42 degrees C. These strains have been used to determine: the kinetics of decay at 30 degrees C of thermally induced ability to enhance UV mutagenesis; the kinetics of thermal enhancement of spontaneous and UV-induced mutability at 42 degrees C, and the kinetics of decay at 30 degrees C of susceptibility to thermal enhancement of spontaneous and UV-induced mutability. Mutations from tryptophane requirement to prototrophy (Trp- to Trp+) were scored. UV doses were 0.2 J/M2 for excision repair-deficient (Uvr-) and 2J/m2 for Uvr+ strains. The results support the following conclusions. 1) thermally induced SOS repair activity decays at 30 degrees C to about 25% of its maximum level in 45 min, and is no longer detectable after 90 min. 2) Thermal enhancement of UV mutability occurs at sites produced primarily (perhaps exclusively) before completion of the first post-irradiation cell division. 3) UV-induced sites susceptible to thermally induced SOS repair are stable at 30 degrees C in cells not containing the error-prone repair system, and are refractory to constitutive error-free repair for at least 2-3 hours. 4) UV produces a potentially mutagenic type of photoproduct in DNA which can, without interacting with another UV lesion, provide a site susceptible to SOS repair, but which is not a sufficient signal for SOS induction. 5) 50-70% of the SOS-mutable SOS-noninducing UV photoproducts are photoreversible pyrimidine dimers. The results are discussed in relation to current models of UV mutagenesis and induction of UV-inducible functions.

DNA Repair in Potorous tridactylus

The DNA synthesized shortly after ultraviolet (UV) irradiation of Potorous tridactylis (PtK) cells sediments more slowly in alkali than that made by nonirradiated cells. The size of the single-strand segments is approximately equal to the average distance between 1 or 2 cyclobutyl pyrimidine dimers in the parental DNA. These data support the notion that dimers are the photoproducts which interrupt normal DNA replication. Upon incubation of irradiated cells the small segments are enlarged to form high molecular weight DNA as in nonirradiated cells. DNA synthesized at long times ( approximately 24 h) after irradiation is made in segments approximately equal to those synthesized by nonirradiated cells, although only 10-15% of the dimers have been removed by excision repair. These data imply that dimers are not the lesions which initially interrupt normal DNA replication in irradiated cells. In an attempt to resolve these conflicting interpretations, PtK cells were exposed to photoreactivating light after irradiation and before pulse-labeling, since photoreactivation repair is specific for only one type of UV lesion. After 1 h of exposure approximately 35% of the pyrimidine dimers have been monomerized, and the reduction in the percentage of dimers correlates with an increased size for the DNA synthesized by irradiated cells. Therefore, we conclude that the dimers are the lesions which initially interrupt DNA replication in irradiated PtK cells. The monomerization of pyrimidine dimers correlates with a disappearance of repair endonuclease-sensitive sites, as measured in vivo immediately after 1 h of photoreactivation, indicating that some of the sites sensitive to the repair endonuclease (from Micrococcus luteus) are pyrimidine dimers. However, at 24 h after irradiation and 1 h of photoreactivation there are no endonuclease-sensitive sites, even though approximately 50% of the pyrimidine dimers remain in the DNA. These data indicate that not all pyrimidine dimers are accessible to the repair endonuclease. The observation that at long times after irradiation DNA is made in segments equal to those synthesized by nonirradiated cells although only a small percentage of the dimers have been removed suggests that an additional repair system alters dimers so that they no longer interrupt DNA replication.

TRANSFER OF PYRIMIDINE DIMERS DURING BACTERIAL CONJUGATION

When male strains of Escherichia coli are irradiated with 254 nm ultraviolet (UV) light and mated with suitable females, DNA is transferred at almost the normal frequency (Howard‐Flanders et al., 1968). Cole (1971) observed that an episome damaged by UV irradiation of the male parent could be transferred to a recipient and restored to activity by administering photoreactivating light. He therefore deduced that UV lesions are transferred to the recipients during bacterial conjugation. The object of this Research Note is to report experiments providing direct evidence of the transfer of pyrimidine dimers ‐the main lesions of UV irradiation.

Constancy of the UV sensitivity of E. coli during the cell cycle.

Abstract— Cells from slowly growing glucose‐limited chemostat cultures of two different strains of Escherichia coli were separated by size on sucrose gradients, and 254‐nm ultraviolet (UV) survival curves were obtained for samples of the different cell sizes. With the repair‐deficient strain Bs‐1 there was no observable change in survival curve during the cell cycle. With the repair‐proficient strain 15 THU, there was no observable change in the final slope, but y‐axis intercepts increased slightly for the older cells. Our results provide further evidence for the approximate independence of UV sensitivity, k, upon cell age within the division cycle and upon the presence or absence of DNA synthesis. They are consistent with a model in which UV lesions in newly replicated strands of DNA do not contribute to lethality.

Nature of the Complementary Strands Synthesized in Vitro upon the Single-Stranded Circular DNA of Bacteriophage øX174 after Ultraviolet Irradiation

This paper describes experiments intended to decide whether UV lesions in DNA act as absolute blocks to chain elongation by the Escherichia coli DNA polymerase or only slow down the polymerization process. Ultraviolet (UV)-irradiated, single-stranded (SS) circular DNA of bacteriophage øX174 was used as template for the polymerase in a reaction mixture in vitro, under conditions allowing synthesis of not more than one complementary strand per template molecule. The mean length of the newly synthesized complementary strands (as determined by velocity sedimentation in alkaline CsCl gradients), as well as the over-all template activity (as measured by deoxyadenosine monophosphate [dAMP] incorporation) was found to decrease with the number of biologically lethal hits sustained by the irradiated templates. With the increase of time or temperature of reaction, the net synthesis of complementary strands increased (as a consequence of increased initiation), but their mean length remained constant. The mean length of synthesized strands was greater than would be expected if all biologically lethal hits were to block the polymerization process. The lethal hits which serve as blocking lesions are inferred to be pyrimidine dimers because it is possible to obtain synthesis of full-length complementary strands if, when heat-denatured, UV-irradiated, double-stranded replicative form (RF II) DNA of bacteriophage øX174 is used as a template, it is pretreated with yeast photoreactivating enzyme (YPRE) in presence of visible light.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes: I. In vitro studies with haemophilus influenzae transforming DNA and yeast photoreactivating enzyme

The photoenzymatic repair of ultraviolet (UV) lesions in DNA is a photochemical reaction which occurs in an enzyme-substrate complex between these lesions and photoreactivating enzyme (PRE). It is shown here that high intensity flash illumination (duration of the order of 1 millisecond) causes this repair in essentially all enzyme-substrate complexes present at the time. This fact allows several kinds of studies. (1) By applying the flash at timed intervals after mixing PRE and UV-irradiated bacterial transforming DNA, the formation of enzyme-substrate complex can be observed directly. The process takes of the order of minutes for its completion at the usual reaction concentrations, and the effect of changing concentration shows that most of this time is required for the extremely dilute reactants (∼ 10 −9 M) to encounter each other in solution. (2) When a sequence of flashes is applied with UV lesions in excess, the resulting stepwise repair permits complex formation to be studied at each level of recovery, from the first set of lesions erased to the last. The result shows that the lesions are heterogeneous with respect to rate of complex formation. (3) The rate of complex formation depends on temperature, but once a comples has been formed at 37° it does not rapidly dissociate on shifting the temperature to 2°. Repeating the experiments at lower light intensity, where not all the complexes present are repaired at each flash, then shows that the actual photochemical process in the complex is temperature-independent over the 2–37° range—at least for the most rapidly complexing lesions. (4) When PRE is allowed to complex with irradiated transforming DNA in the dark, and irradiated non-transforming DNA is subsequently added flashes applied at various times after this addition allow the dark dissociation of enzyme-substrate complexes to be followed. Comparison with the converse experiment, in which PRE is first complexed with lesions on non-transforming DNA, shows that some complexes are extremely stable, the enzyme preferentially remaining on the first DNA with which it forms a complex even after 2 h. Complexes with UV lesions on the synthetic polydeoxynucleotides dA:dT and dG:dC are more stable than most of complexes with natural DNA. (5) Measurement of complex formation under conditions of DNA excess, where essentially all the enzyme is bound, permits determination of the number of enzyme molecules relative to the number of UV lesions. The reasonable assumption that the number of lesions equals the number of pyrimidine dimers recoverable from the DNA then gives an absolute count. Provided the molecular weight of 3·10 4 given by Muhammed is approximately correct, the proportion of pure enzyme to totat protein in crude yeast extract is about 2·10 −6.