In vivo reduction of RAD51-mediated homologous recombination triggers aging but impairs oncogenesis.

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.

DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability.

The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.

Suleiman-El-Hattab syndrome: a histone modification disorder caused by TASP1 deficiency.

BACKGROUND: TASP1 encodes an endopeptidase activating histone methyltransferases of the KMT2 family. Homozygous loss-of-function variants in TASP1 have recently been associated with Suleiman-El-Hattab syndrome. We report six individuals with Suleiman-El-Hattab syndrome and provide functional characterization of this novel histone modification disorder in a multi-omics approach. METHODS: Chromosomal microarray/exome sequencing in all individuals. Western blotting from fibroblasts in two individuals. RNA sequencing and proteomics from fibroblasts in one individual. Methylome analysis from blood in two individuals. Knock-out of tasp1 orthologue in zebrafish and phenotyping. RESULTS: All individuals had biallelic TASP1 loss-of-function variants and a phenotype including developmental delay, multiple congenital anomalies (including cardiovascular and posterior fossa malformations), a distinct facial appearance and happy demeanor. Western blot revealed absence of TASP1. RNA sequencing/proteomics showed HOX gene downregulation (HOXA4, HOXA7, HOXA1 and HOXB2) and dysregulation of transcription factor TFIIA. A distinct methylation profile intermediate between control and Kabuki syndrome (KMT2D) profiles could be produced. Zebrafish tasp1 knock-out revealed smaller head size and abnormal cranial cartilage formation in tasp1 crispants. CONCLUSION: This work further delineates Suleiman-El-Hattab syndrome, a recognizable neurodevelopmental syndrome. Possible downstream mechanisms of TASP1 deficiency include perturbed HOX gene expression and dysregulated TFIIA complex. Methylation pattern suggests that Suleiman-El-Hattab syndrome can be categorized into the group of histone modification disorders including Wiedemann-Steiner and Kabuki syndrome.

Changes in the architecture and abundance of replication intermediates delineate the chronology of DNA damage tolerance pathways at UV-stalled replication forks in human cells.

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.

The SLX4 complex is a SUMO E3 ligase that impacts on replication stress outcome and genome stability.

The SLX4 Fanconi anemia protein is a tumor suppressor that may act as a key regulator that engages the cell into specific genome maintenance pathways. Here, we show that the SLX4 complex is a SUMO E3 ligase that SUMOylates SLX4 itself and the XPF subunit of the DNA repair/recombination XPF-ERCC1 endonuclease. This SLX4-dependent activity is mediated by a remarkably specific interaction between SLX4 and the SUMO-charged E2 conjugating enzyme UBC9 and relies not only on newly identified SUMO-interacting motifs (SIMs) in SLX4 but also on its BTB domain. In contrast to its ubiquitin-binding UBZ4 motifs, SLX4 SIMs are dispensable for its DNA interstrand crosslink repair functions. Instead, while detrimental in response to global replication stress, the SUMO E3 ligase activity of the SLX4 complex is critical to prevent mitotic catastrophe following common fragile site expression.

First description of ultramutated endometrial cancer caused by germline loss-of-function and somatic exonuclease domain mutations in POLE gene.

Endometrial cancer (EC) harboring heterozygous POLE proofreading inactivating mutations (POLE-exo*) is associated with an increased number of somatic mutations that result in a distinctive anti-tumor immune response. However, the consequences of such POLE mutations in the context of the missing wild-type allele have not yet been described in endometrial tumors. A 72-year-old woman harboring a germline monoallelic frameshift mutation (p.Pro269fsTer26) in POLE was diagnosed with an EC having a somatic heterozygous mutation in the exonuclease domain of POLE (S459F). Targeted gene sequencing revealed an ultramutated phenotype (381 mutations/Mb) in the tumor and a 2-fold excess of mutations on the DNA leading strand. Additionally, we observed a mutational signature similar to the COSMIC signature 10, a higher mutation rate in this tumor than in endometrial tumors with heterozygous POLE-exo*, and an increased number of T lymphocytes. This is the first report of an ultramutated EC harboring a somatic POLE-exo* mutation in association with a germline loss-of-function mutation in this gene. The absence of a wild type POLE allele led to a particularly high mutational burden.

The hMsh2-hMsh6 complex acts in concert with monoubiquitinated PCNA and Pol η in response to oxidative DNA damage in human cells.

Posttranslational modification of PCNA by ubiquitin plays an important role in coordinating the processes of DNA damage tolerance during DNA replication. The monoubiquitination of PCNA was shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta (η) to bypass UV-induced DNA lesions during replication. Here, we show that in response to oxidative stress, PCNA becomes transiently monoubiquitinated in an S phase- and USP1-independent manner. Moreover, Polη interacts with mUb-PCNA at sites of oxidative DNA damage via its PCNA-binding and ubiquitin-binding motifs. Strikingly, while functional base excision repair is not required for this modification of PCNA or Polη recruitment to chromatin, the presence of hMsh2-hMsh6 is indispensable. Our findings highlight an alternative pathway in response to oxidative DNA damage that may coordinate the removal of oxidatively induced clustered DNA lesions and could explain the high levels of oxidized DNA lesions in MSH2-deficient cells.

PUB-NChIP--"in vivo biotinylation" approach to study chromatin in proximity to a protein of interest.

We have developed an approach termed PUB-NChIP (proximity utilizing biotinylation with native ChIP) to purify and study the protein composition of chromatin in proximity to a nuclear protein of interest. It is based on coexpression of (1) a protein of interest, fused with the bacterial biotin ligase BirA, together with (2) a histone fused to a biotin acceptor peptide (BAP), which is specifically biotinylated by BirA-fusion in the proximity of the protein of interest. Using the RAD18 protein as a model, we demonstrate that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2AZ has a different pattern of H4 acetylation. Finally, biotin pulse-chase experiments show that the H4 acetylation pattern starts to resemble the acetylation pattern of total H4 after the proximity of chromatin to RAD18 has been lost.

The helicase FBH1 is tightly regulated by PCNA via CRL4(Cdt2)-mediated proteolysis in human cells.

During replication, DNA damage can challenge replication fork progression and cell viability. Homologous Recombination (HR) and Translesion Synthesis (TLS) pathways appear as major players involved in the resumption and completion of DNA replication. How both pathways are coordinated in human cells to maintain genome stability is unclear. Numerous helicases are involved in HR regulation. Among them, the helicase FBH1 accumulates at sites of DNA damage and potentially constrains HR via its anti-recombinase activity. However, little is known about its regulation in vivo. Here, we report a mechanism that controls the degradation of FBH1 after DNA damage. Firstly, we found that the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is critical for FBH1 recruitment to replication factories or DNA damage sites. We then showed the anti-recombinase activity of FBH1 is partially dependent on its interaction with PCNA. Intriguingly, after its re-localization, FBH1 is targeted for degradation by the Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2))-PCNA pathway via a PCNA-interacting peptide (PIP) degron. Importantly, expression of non-degradable FBH1 mutant impairs the recruitment of the TLS polymerase eta to chromatin in UV-irradiated cells. Thus, we propose that after DNA damage, FBH1 might be required to restrict HR and then degraded by the Cdt2-proteasome pathway to facilitate TLS pathway.

Correlation of phenotype/genotype in a cohort of 23 xeroderma pigmentosum-variant patients reveals 12 new disease-causing POLH mutations.

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XPV patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.

Genomic mutation landscape of skin cancers from DNA repair-deficient xeroderma pigmentosum patients.

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyze 38 skin cancer genomes from five XP groups. We find that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH knockout cell line reveal the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3' nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.

Trastuzumab deruxtecan in metastatic breast cancer with variable HER2 expression: the phase 2 DAISY trial.

The mechanisms of action of and resistance to trastuzumab deruxtecan (T-DXd), an anti-HER2-drug conjugate for breast cancer treatment, remain unclear. The phase 2 DAISY trial evaluated the efficacy of T-DXd in patients with HER2-overexpressing (n = 72, cohort 1), HER2-low (n = 74, cohort 2) and HER2 non-expressing (n = 40, cohort 3) metastatic breast cancer. In the full analysis set population (n = 177), the confirmed objective response rate (primary endpoint) was 70.6% (95% confidence interval (CI) 58.3-81) in cohort 1, 37.5% (95% CI 26.4-49.7) in cohort 2 and 29.7% (95% CI 15.9-47) in cohort 3. The primary endpoint was met in cohorts 1 and 2. Secondary endpoints included safety. No new safety signals were observed. During treatment, HER2-expressing tumors (n = 4) presented strong T-DXd staining. Conversely, HER2 immunohistochemistry 0 samples (n = 3) presented no or very few T-DXd staining (Pearson correlation coefficient r = 0.75, P = 0.053). Among patients with HER2 immunohistochemistry 0 metastatic breast cancer, 5 of 14 (35.7%, 95% CI 12.8-64.9) with ERBB2 expression below the median presented a confirmed objective response as compared to 3 of 10 (30%, 95% CI 6.7-65.2) with ERBB2 expression above the median. Although HER2 expression is a determinant of T-DXd efficacy, our study suggests that additional mechanisms may also be involved. (ClinicalTrials.gov identifier NCT04132960 .).

ATR/Chk1 pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells.

DNA polymerase eta (poleta) performs translesion synthesis past ultraviolet (UV) photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XP-V) syndrome. The slight sensitivity of XP-V cells to UV is dramatically enhanced by low concentrations of caffeine. So far, the biological explanation for this feature remains elusive. Using DNA combing, we showed that translesion synthesis defect leads to a strong reduction in the number of active replication forks and a high proportion of stalled forks in human cells, which contrasts with budding yeast. Moreover, extensive regions of single-strand DNA are formed during replication in irradiated XP-V cells, leading to an over-activation of ATR/Chk1 pathway after low UVC doses. Addition of a low concentration of caffeine post-irradiation, although inefficient to restore S-phase progression, significantly decreases Chk1 activation and abrogates DNA synthesis in XP-V cells. While inhibition of Chk1 activity by UCN-01 prevents UVC-induced S-phase delay in wild-type cells, it aggravates replication defect in XP-V cells by increasing fork stalling. Consequently, UCN-01 sensitizes XP-V cells to UVC as caffeine does. Our findings indicate that poleta acts at stalled forks to resume their progression, preventing the requirement for efficient replication checkpoint after low UVC doses. In the absence of poleta, Chk1 kinase becomes essential for replication resumption by alternative pathways, via fork stabilization.

Human DNA polymerase iota protects cells against oxidative stress.

Human DNA polymerase iota (poliota) is a unique member of the Y-family of specialised polymerases that displays a 5'deoxyribose phosphate (dRP) lyase activity. Although poliota is well conserved in higher eukaryotes, its role in mammalian cells remains unclear. To investigate the biological importance of poliota in human cells, we generated fibroblasts stably downregulating poliota (MRC5-pol iota(KD)) and examined their response to several types of DNA-damaging agents. We show that cell lines downregulating poliota exhibit hypersensitivity to DNA damage induced by hydrogen peroxide (H(2)O(2)) or menadione but not to ethylmethane sulphonate (EMS), UVC or UVA. Interestingly, extracts from cells downregulating poliota show reduced base excision repair (BER) activity. In addition, poliota binds to chromatin after treatment of cells with H(2)O(2) and interacts with the BER factor XRCC1. Finally, green fluorescent protein-tagged poliota accumulates at the sites of oxidative DNA damage in living cells. This recruitment is partially mediated by its dRP lyase domain and ubiquitin-binding domains. These data reveal a novel role of human poliota in protecting cells from oxidative damage.

Predicting immunotherapy outcomes in patients with MSI tumors using NLR and CT global tumor volume.

Background: Anti-PD-(L)1 treatment is indicated for patients with mismatch repair-deficient (MMRD) tumors, regardless of tumor origin. However, the response rate is highly heterogeneous across MMRD tumors. The objective of the study is to find a score that predicts anti-PD-(L)1 response in patients with MMRD tumors. Methods: Sixty-one patients with various origin of MMRD tumors and treated with anti-PD-(L)1 were retrospectively included in this study. An expert radiologist annotated all tumors present at the baseline and first evaluation CT-scans for all the patients by circumscribing them on their largest axial axis (single slice), allowing us to compute an approximation of their tumor volume. In total, 2120 lesions were annotated, which led to the computation of the total tumor volume for each patient. The RECIST sum of target lesions' diameters and neutrophile-to-lymphocyte (NLR) were also reported at both examinations. These parameters were determined at baseline and first evaluation and the variation between the first evaluation and baseline was calculated, to determine a comprehensive score for overall survival (OS) and progression-free survival (PFS). Results: Total tumor volume at baseline was found to be significantly correlated to the OS (p-value: 0.005) and to the PFS (p-value:<0.001). The variation of the RECIST sum of target lesions' diameters, total tumor volume and NLR were found to be significantly associated to the OS (p-values:<0.001, 0.006,<0.001 respectively) and to the PFS (<0.001,<0.001, 0.007 respectively). The concordance score combining total tumor volume and NLR variation was better at stratifying patients compared to the tumor volume or NLR taken individually according to the OS (pairwise log-rank test p-values: 0.033,<0.001, 0.002) and PFS (pairwise log-rank test p-values: 0.041,<0.001, 0.003). Conclusion: Total tumor volume appears to be a prognostic biomarker of anti-PD-(L)1 response to immunotherapy in metastatic patients with MMRD tumors. Combining tumor volume and NLR with a simple concordance score stratifies patients well according to their survival and offers a good predictive measure of response to immunotherapy.

Targeting genome integrity dysfunctions impedes metastatic potency in non-small cell lung cancer circulating tumor cell-derived explants.

DNA damage and genomic instability contribute to non-small cell lung cancer (NSCLC) etiology and progression. However, their therapeutic exploitation is disappointing. CTC-derived explants (CDX) offer systems for mechanistic investigation of CTC metastatic potency and may provide rationale for biology-driven therapeutics. Four CDX models and 3 CDX-derived cell lines were established from NSCLC CTCs and recapitulated patient tumor histology and response to platinum-based chemotherapy. CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4) demonstrated considerable mutational landscape similarity with patient tumor biopsy and/or single CTCs. Truncal alterations in key DNA damage response (DDR) and genome integrity-related genes were prevalent across models and assessed as therapeutic targets in vitro, in ovo, and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to biallelic BRCA2 mutation and FANCA deletion, unrepaired DNA lesions after mitosis, and olaparib sensitivity, despite resistance to chemotherapy. SLFN11 overexpression in GR-CDXL4 led to olaparib sensitivity and was in coherence with neuroendocrine marker expression in patient tumor biopsy, suggesting a predictive value of SLFN11 in NSCLC histological transformation into small cell lung cancer (SCLC). Centrosome clustering promoted targetable chromosomal instability in GR-CDXL3 cells. These CDX unravel DDR and genome integrity-related defects as a central mechanism underpinning metastatic potency of CTCs and provide rationale for their therapeutic targeting in metastatic NSCLC.

Overcoming resistance to αPD-1 of MMR-deficient tumors with high tumor-induced neutrophils levels by combination of αCTLA-4 and αPD-1 blockers.

BACKGROUND: Clinical studies have highlighted the efficacy of anti-programmed death 1 (αPD-1) monoclonal antibodies in patients with DNA mismatch repair-deficient (MMRD) tumors. However, the responsiveness of MMRD cancers to αPD-1 therapy is highly heterogeneous, and the origins of this variability remain not fully understood. METHODS: 4T1 and CT26 mouse tumor cell lines were inactivated for the MMRD gene Msh2, leading to a massive accumulation of mutations after serial passages of cells. Insertions/deletion events and mutation load were evaluated by whole exome sequencing. Mice bearing highly mutated MMRD tumor or parental tumors were treated with αPD-1 and tumor volume was monitored. Immune cell type abundance was dynamically assessed in the tumor microenvironment and the blood by flow cytometry. Neutrophils were depleted in mice using αLY6G antibody, and regulatory T (Treg) cell population was reduced with αCD25 or anti-cytotoxic T-lymphocytes-associated protein 4 (αCTLA-4) antibodies. Patients with MMRD tumors treated with immune checkpoint blockade-based therapy were retrospectively identified and neutrophil-to-lymphocyte ratio (NLR) was evaluated and examined for correlation with clinical benefit. RESULTS: By recapitulating mismatch repair deficiency in different mouse tumor models, we revealed that elevated circulating tumor-induced neutrophils (TIN) in hypermutated MMRD tumors hampered response to αPD-1 monotherapy. Importantly, depletion of TIN using αLy-6G antibody reduced Treg cells and restored αPD-1 response. Conversely, targeting Treg cells by αCD25 or αCTLA-4 antibodies limited peripheral TIN accumulation and elicited response in αPD-1-resistant MMRD tumors, highlighting a crosstalk between TIN and Treg cells. Thus, αPD-1+αCTLA-4 combination overcomes TIN-induced resistance to αPD-1 in mice bearing MMRD tumors. Finally, in a cohort of human (high microsatellite instability)/MMRD tumors we revealed that early on-treatment change in the NLR ratio may predict resistance to αPD-1 therapy. CONCLUSIONS: TIN countered αPD-1 efficacy in MMRD tumors. Since αCTLA-4 could restrict TIN accumulation, αPD-1+αCTLA-4 combination overcomes αPD-1 resistance in hosts with hypermutated MMRD tumors displaying abnormal neutrophil accumulation.

PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo.

The common techniques to study protein-protein proximity in vivo are not well adapted to the capabilities and the expertise of a standard proteomics laboratory, typically based on the use of mass spectrometry. With the aim of closing this gap, we have developed PUB-MS (for proximity utilizing biotinylation and mass spectrometry), an approach to monitor protein-protein proximity, based on biotinylation of a protein fused to a biotin-acceptor peptide (BAP) by a biotin-ligase, BirA, fused to its interaction partner. The biotinylation status of the BAP can be further detected by either Western analysis or mass spectrometry. The BAP sequence was redesigned for easy monitoring of the biotinylation status by LC-MS/MS. In several experimental models, we demonstrate that the biotinylation in vivo is specifically enhanced when the BAP- and BirA-fused proteins are in proximity to each other. The advantage of mass spectrometry is demonstrated by using BAPs with different sequences in a single experiment (allowing multiplex analysis) and by the use of stable isotopes. Finally, we show that our methodology can be also used to study a specific subfraction of a protein of interest that was in proximity with another protein at a predefined time before the analysis.

Regulation of proliferating cell nuclear antigen ubiquitination in mammalian cells.

After exposure to DNA-damaging agents that block the progress of the replication fork, monoubiquitination of proliferating cell nuclear antigen (PCNA) mediates the switch from replicative to translesion synthesis DNA polymerases. We show that in human cells, PCNA is monoubiquitinated in response to methyl methanesulfonate and mitomycin C, as well as UV light, albeit with different kinetics, but not in response to bleomycin or camptothecin. Cyclobutane pyrimidine dimers are responsible for most of the PCNA ubiquitination events after UV-irradiation. Failure to ubiquitinate PCNA results in substantial sensitivity to UV and methyl methanesulfonate, but not to camptothecin or bleomycin. PCNA ubiquitination depends on Replication Protein A (RPA), but is independent of ATR-mediated checkpoint activation. After UV-irradiation, there is a temporal correlation between the disappearance of the deubiquitinating enzyme USP1 and the presence of PCNA ubiquitination, but this correlation was not found after chemical mutagen treatment. By using cells expressing photolyases, we are able to remove the UV lesions, and we show that PCNA ubiquitination persists for many hours after the damage has been removed. We present a model of translesion synthesis behind the replication fork to explain the persistence of ubiquitinated PCNA.