DNA damage induced Pol η recruitment takes place independently of the cell cycle phase

When DNA is damaged in cells progressing through S phase, replication blockage can be avoided by TLS (Translesion DNA synthesis). This is an auxiliary replication mechanism that relies on the function of specialized polymerases that accomplish DNA damage bypass. Intriguingly, recent evidence has linked TLS polymerases to processes that can also take place outside S phase such as nucleotide excision repair (NER). Here we show that Pol η is recruited to UV-induced DNA lesions in cells outside S phase including cells permanently arrested in G1. This observation was confirmed by different strategies including global UV irradiation, local UV irradiation and local multi-photon laser irradiation of single nuclei in living cells. The potential connection between Pol η recruitment to DNA lesions outside S phase and NER was further evaluated. Interestingly, the recruitment of Pol η to damage sites outside S phase did not depend on active NER, as UV-induced focus formation occurred normally in XPA, XPG and XPF deficient fibroblasts. Our data reveals that the re-localization of the TLS polymerase Pol η to photo-lesions might be temporally and mechanistically uncoupled from replicative DNA synthesis and from DNA damage processing.     

New functions for Y family polymerases

The Y family of DNA polymerases plays crucial roles in carrying out translesion synthesis past damaged bases in DNA. Several recent papers suggest that they might have other roles as well in gene conversion, in nucleotide excision repair (NER), and in DNA replication under stressed conditions.     

Suffering in silence: the tolerance of DNA damage

When cells that are actively replicating DNA encounter sites of base damage or strand breaks, replication might stall or arrest. In this situation, cells rely on DNA-damage-tolerance mechanisms to bypass the damage effectively. One of these mechanisms, known as translesion DNA synthesis, is supported by specialized DNA polymerases that are able to catalyse nucleotide incorporation opposite lesions that cannot be negotiated by high-fidelity replicative polymerases. A second category of tolerance mechanism involves alternative replication strategies that obviate the need to replicate directly across sites of template-strand damage.     

The Y-family DNA polymerase kappa (pol kappa) functions in mammalian nucleotide-excision repair

DNA polymerase kappa (pol kappa) is a member of the Y-family of DNA polymerases that are thought to function in translesion synthesis (TLS) past different types of DNA damage. Here, we show that pol kappa-deficient mouse cells have substantially reduced (but not absent) levels of nucleotide excision repair (NER) of UV damage, as measured by several methods. Our results provide evidence for an unexpected role for pol kappa in mammalian NER.     

The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes

AA8 Chinese hamster ovary cells were treated with halogenated nucleosides analogues of thymidine, namely CldU, 5-iodo-2'-deoxyuridine (IdU), and 5-bromo-2'-deoxyuridine (BrdU), following different experimental protocols. The purpose was to see whether incorporation of exogenous pyrimidine analogues into DNA could interfere with normal chromosome segregation. The endpoint chosen was endoreduplication, that arises after aberrant mitosis when daughter chromatids segregation fails. Treatment with any of the halogenated nucleosides for two consecutive cell cycles resulted in endoreduplication, with a highest yield for CldU, intermediate for IdU, and lowest for BrdU. The frequency of endoreduplicated cells paralleled in all cases the level of analogue substitution into DNA. Our results seem to support that thymidine analogue substitution into DNA is responsible for the triggering of endoreduplication. Besides, the lack of any effect on endoreduplication when CldU was present for only one S-period strongly suggest that it is the nature of template, and not nascent DNA, that plays a major role in chromosome segregation. Taking into account that topoisomerase II cleaves DNA at preferred sequences within its recognition/binding sites, the likely involvement of the enzyme is discussed.     

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed ‘unscheduled DNA synthesis (UDS)’, is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.     

DDB2-Independent Role for p53 in the Recovery from Ultraviolet Light-Induced Replication Arrest

Ultraviolet light (UV light) induces helix distorting DNA lesions that pose a block to replicative DNA polymerases. Recovery from this replication arrest is reportedly impaired in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) fibroblasts and primary fibroblasts lacking functional p53. These independent observations suggested that the involvement of p53 in the recovery from UV-induced replication arrest was related to its role in regulating the global genomic subpathway of NER (GG-NER). Using primary human fibroblasts, we confirm that the recovery from UV-induced replication arrest is impaired in cells lacking functional p53 and in primary XP fibroblasts derived from complementation groups A or C (XP-A and XP-C) that are defective in GG-NER. Surprisingly, DNA synthesis recovered normally in GG-NER-deficient XP complementation group E (XP-E) cells that carry mutations in the p53 regulated DNA repair gene DDB2 and are specifically defective in the repair of cyclobutane pyrimidine dimers (CPD) but not pyrimidine (6-4) pyrimidone photoproducts. Disruption of p53 in these XP-E fibroblasts prevented the recovery from UV-induced replication arrest. Therefore, the roles of p53 and GG-NER in the recovery from UV-induced replication are separable and DDB2-independent. These results further indicate that primary human fibroblasts expressing functional p53 efficiently replicate DNA containing CPD whereas p53-deficient cells do not, consistent with a role for p53 in permitting translesion DNA synthesis of these DNA lesions.     

Fine structural in situ analysis of nascent DNA movement following DNA replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase alpha was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

An evolving tail of centromere histone variant CENP-A

We have recently shown that replication forks pause near origins in normal human fibroblasts (NHF1-hTERT) but not glioblastoma T98G cells. This observation led us to question whether other differences in the replication program may exist between these cell types that may relate to their genetic integrity. To identify differences, we detected immunoflourescently the sequential incorporation of the nucleotide analogs IdU and CldU into replicating DNA at the start of every hour of a synchronized S phase. We then characterized the patterns of labeled replicating DNA tracks and quantified the percentages and lengths of the tracks found at these hourly intervals. From the directionality of labeling in single extended replicating DNA fibers, tracks were categorized as single bidirectional origins, unidirectional elongations, clusters of origins firing in tandem, or merging forks (terminations). Our analysis showed that the start of S phase is enriched in single bidirectional origins in NHF1-hTERT cells, followed by an increase in clustering during mid S phase and an increase in merging forks during late S phase. Early S phase in T98G cells also largely consisted of single bidirectional origin initiations; however, an increase in clustering was delayed until an hour later, and clusters were shorter in mid/late S phase than in NHF1-hTERT cells. The spike in merging forks also did not occur until an hour later in T98G cells. Our observations suggest models to explain the temporal replication of single and clustered origins, and suggest differences in the replication program in a normal and cancer cell line.     

Polymerase zeta dependency of increased adaptive mutation frequencies in nucleotide excision repair-deficient yeast strains

Reversions of an auxotrophy-causing frameshift allele during prolonged starvation of yeast cells were used as a means to elucidate the mechanisms concerned with the generation of spontaneous adaptive mutations in cell cycle-arrested cells. Whereas about 50% of these reversions were previously shown to depend on the non-homologous end joining pathway of DNA double-strand break repair, the origin of the residual 50% remains unknown. In search for a mechanism for generation of the latter fraction of reversions we examined the role of the translesion synthesis (TLS) polymerases zeta, eta and Rev1p in cells with wild-type or impaired nucleotide excision repair (NER) capacity. The basal level of adaptive mutations in the repair-proficient wild type was not influenced by disruptions of the genes coding for these three TLS polymerases. Intriguingly, a deficiency in NER by disruption of RAD14, RAD16 or RAD26 resulted in a significantly higher frequency of adaptive mutation, yet this increase was strictly dependent on an intact REV3 gene, coding for the catalytic subunit of polymerase zeta. Furthermore, we observed that intact REV3 was also required for the occurrence of increased frequencies of adaptive mutants in the NER-proficient wild type following UV irradiation. While in proliferating cells the translesion synthesis function of polymerase zeta is connected to DNA replication, our data suggest that in cell cycle-arrested cells this enzyme is able to carry out either TLS or error-prone polymerization along an undamaged template in the course of repair processes. Such a hitherto unappreciated activity of polymerase zeta in non-replicating cells may contribute to the incidence of mutations in evolution, aging and cancer.     

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA.     

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase α was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.     

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21(Cip1, Waf1, Sdi1) is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase delta. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.     

A single domain in human DNA polymerase iota mediates interaction with PCNA: implications for translesion DNA synthesis

DNA polymerases (Pols) of the Y family rescue stalled replication forks by promoting replication through DNA lesions. Humans have four Y family Pols, eta, iota, kappa, and Rev1, of which Pols eta, iota, and kappa have been shown to physically interact with proliferating cell nuclear antigen (PCNA) and be functionally stimulated by it. However, in sharp contrast to the large increase in processivity that PCNA binding imparts to the replicative Pol, Poldelta, the processivity of Y family Pols is not enhanced upon PCNA binding. Instead, PCNA binding improves the efficiency of nucleotide incorporation via a reduction in the apparent K(m) for the nucleotide. Here we show that Poliota interacts with PCNA via only one of its conserved PCNA binding motifs, regardless of whether PCNA is bound to DNA or not. The mode of PCNA binding by Poliota is quite unlike that in Poldelta, where multisite interactions with PCNA provide for a very tight binding of the replicating Pol with PCNA. We discuss the implications of these observations for the accuracy of DNA synthesis during translesion synthesis and for the process of Pol exchange at the lesion site.     

Caffeine delays replication fork progression and enhances UV-induced homologous recombination in Chinese hamster cell lines

The ability to bypass DNA lesions encountered during replication is important in order to maintain cell viability and avoid genomic instability. Exposure of mammalian cells to UV-irradiation induces the formation of DNA lesions that stall replication forks. In order to restore replication, different bypass mechanisms are operating, previously named post-replication repair. Translesion DNA synthesis is performed by low-fidelity polymerases, which can replicate across damaged sites. The nature of lesions and of polymerases involved influences the resulting frequency of mutations. Homologous recombination represents an alternative pathway for the rescue of stalled replication forks. Caffeine has long been recognized to influence post-replication repair, although the mechanism is not identified. Here, we found that caffeine delays the progress of replication forks in UV-irradiated Chinese hamster cells. The length of this enhanced delay was similar in wild-type cells and in cell deficient in either homologous recombination or nucleotide excision repair. Furthermore, caffeine attenuated the frequency of UV-induced mutations in the hprt gene, whereas the frequency of recombination, monitored in this same gene, was enhanced. These observations indicate that in cells exposed to UV-light, caffeine inhibits the rescue of stalled replication forks by translesion DNA synthesis, thereby causing a switch to bypass via homologous recombination. The biological consequence of the former pathway is mutations, while the latter results in chromosomal aberrations.     

Gap-filling and bypass at the replication fork are both active mechanisms for tolerance of low-dose ultraviolet-induced DNA damage in the human genome

Ultraviolet (UV)-induced DNA damage are removed by nucleotide excision repair (NER) or can be tolerated by specialized translesion synthesis (TLS) polymerases, such as Polη. TLS may act at stalled replication forks or through an S-phase independent gap-filling mechanism. After UVC irradiation, Polη-deficient (XP-V) human cells were arrested in early S-phase and exhibited both single-strand DNA (ssDNA) and prolonged replication fork stalling, as detected by DNA fiber assay. In contrast, NER deficiency in XP-C cells caused no apparent defect in S-phase progression despite the accumulation of ssDNA and a G2-phase arrest. These data indicate that while Polη is essential for DNA synthesis at ongoing damaged replication forks, NER deficiency might unmask the involvement of tolerance pathway through a gap-filling mechanism. ATR knock down by siRNA or caffeine addition provoked increased cell death in both XP-V and XP-C cells exposed to low-dose of UVC, underscoring the involvement of ATR/Chk1 pathway in both DNA damage tolerance mechanisms. We generated a unique human cell line deficient in XPC and Polη proteins, which exhibited both S- and G2-phase arrest after UVC irradiation, consistent with both single deficiencies. In these XP-C/Polη^KD cells, UVC-induced replicative intermediates may collapse into double-strand breaks, leading to cell death. In conclusion, both TLS at stalled replication forks and gap-filling are active mechanisms for the tolerance of UVC-induced DNA damage in human cells and the preference for one or another pathway depends on the cellular genotype.     

UV-induced immobilization of DNA repair protein XPA in different human cell line

XPA repair protein is absolutely needed for nucleotide excision repair (NER). It preferentially binds UV-irradiated DNA in vitro and possibly takes place in the recognition of pyrimidine dimers, the main type of UV-lesions in DNA. Using immunofluorescent microscopy and immunoblotting technique we have found that XPA protein is fully extractable by Triton X-100 solution from non-irradiated normal human fibroblasts, but after UV-irradiation its extractability decreases in UV-dose dependent manner. UV-induced XPA-immobilization was observed in human cell lines with different types of repair defects, but XPA-extractability from unirradiated cells of these lines was significantly lower in comparison with normal fibroblasts. These data do not permit to make conclusion concerning the distinct connection of this phenomenon with different pathways of NER. Histone deacetylase inhibitor, sodium butyrate, did not change the level of extractability in unirradiated and UV-irradiated normal human cells and CHO cells, defective in global genome repair, that indicated the independence of XPA-immobilization from the level of histone acetylation. It was established with the help of confocal microscopy that XPA-foci in detergent-treated UV-irradiated cell were partially colocalized with the focal sites of PCNA, an auxiliary protein of DNA polymerases delta and epsilon. It may mean that a part of detergent-resistant XPA foci correspond to DNA repair synthesis sites, but the major part of immobilized XPA reflects the early step of repair proteins assembly formation needed for the repair of the lesions.     

In situ enzymology of DNA replication and ultraviolet-induced DNA repair synthesis in permeable human cells

Using permeable diploid human fibroblasts, we have studied the deoxyribonucleoside triphosphate concentration dependences of ultraviolet- (UV-) induced DNA repair synthesis and semiconservative DNA replication. In both cell types (AG1518 and IMR-90) examined, the apparent Km values for dCTP, dGTP, and dTTP for DNA replication were between 1.2 and 2.9 microM. For UV-induced DNA repair synthesis, the apparent Km values were substantially lower, ranging from 0.11 to 0.44 microM for AG1518 cells and from 0.06 to 0.24 microM for IMR-90 cells. Control experiments established that these values were not significantly influenced by nucleotide degradation during the permeable cell incubations or by the presence of residual endogenous nucleotides within the permeable cells. Recent data implicate DNA polymerase delta in UV-induced repair synthesis and suggest that DNA polymerases alpha and delta are both involved in semiconservative replication. We measured Km values for dGTP and dTTP for polymerases alpha and delta, for comparison with the values for replication and repair synthesis. Km values for polymerase alpha were 2.0 microM for dGTP and 5.0 microM for dTTP. For polymerase delta, the Km values were 2.0 microM for dGTP and 3.5 microM for dTTP. The deoxyribonucleotide Km values for DNA polymerase delta are much greater than the Km values for UV-induced repair synthesis, suggesting that when polymerase delta functions in DNA repair, its characteristics are altered substantially either by association with accessory proteins or by direct posttranslational modification. In contrast, the deoxyribonucleotide binding characteristics of the DNA replication machinery differ little from those of the isolated DNA polymerases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3^−/− and Rev1^−/− cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3^−/− and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.