Deep intronic founder mutations identified in the ERCC4/XPF gene are potential therapeutic targets for a high-frequency form of xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a genodermatosis defined by cutaneous photosensitivity with an increased risk of skin tumors because of DNA repair deficiency. The worldwide prevalence of XP is ~1 to 4 in million, with higher incidence in some countries and regions including Japan (1 in 22,000) and North Africa due to founder mutations and a high degree of consanguinity. Among XP, the complementation group F (XP-F), is a rare form (1% of worldwide XP); however, this is underdiagnosed, because the ERCC4/XPF gene is essential for fetal development and most of previously reported ERCC4/XPF pathogenic variants are hypomorphs causing relatively mild phenotypes. From the largest Japanese XP cohort study, we report 17 XP-F cases bearing two pathogenic variants, both identified in deep intronic regions of the ERCC4/XPF gene. The first variant, located in intron 1, is a Japanese founder mutation, which additionally accounts for ~10% of the entire Japanese XP cases (MAF = 0.00196), causing an aberrant pre-mRNA splicing due to a miss-binding of U1snRNA. The second mutation located in intron eight induces an alternative polyadenylation. Both mutations cause a reduction of the ERCC4/XPF gene expression, resulting in XP clinical manifestations. Most cases developed early-onset skin cancers, indicating that these variants need critical attention. We further demonstrate that antisense oligonucleotides designed for the mutations can restore the XPF protein expression and DNA repair capacity in the patients' cells. Collectively, these pathogenic variants can be potential therapeutic targets for XP.

Association of combined hip-pelvic-lumbar mobility with hip muscle strength and clinical outcomes in patients treated for femoroacetabular impingement syndrome: A case-control study.

BACKGROUND: It is unclear whether hip and pelvic mobility in the sagittal plane are associated with hip function in FAIS. This study aimed to determine whether hip-pelvis-lumbar mobility is associated with preoperative hip function and postoperative outcomes in FAIS. METHODS: This was a level 3 case-control study. This study included 111 patients who underwent arthroscopic FAI correction and labral preservation between 2015 and 2019. The Hip-Pelvic-Lumbar Mobility Test (HPLMT) was performed preoperatively; hip flexion with the hip adducted and internally rotated was examined in the lateral decubitus position, and a total hip flexion angle of less than 120° was diagnosed as positive. HPLMT-positive patients were classified as cases, and HPLMT-negative patients were classified as controls. Hip muscle strength was measured preoperatively using a hand-held dynamometer. The modified Harris hip score (mHHS), Nonarthritic Hip Score (NAHS), and International Hip Outcome Tool-12 score were obtained preoperatively and postoperatively. The rates of patient acceptable symptomatic state (PASS) achievement for patient-reported outcome scores (PROSs) were compared between groups. RESULTS: The preoperative PROSs for the HPLMT-positive patients were significantly lower than those for the HPLMT-negative patients. HPLMT-positive patients had significantly weaker hip strength on the affected side than HPLMT-negative patients (flexion; p < 0.001, abduction; p = 0.001). HPLMT-positive patients had significantly lower postoperative mHHS than HPLMT-negative patients. HPLMT-positive patients were significantly less likely to achieve a PASS for the mHHS (62% versus 85%) and NAHS (48% versus 71%) than HPLMT-negative patients. CONCLUSION: Hip-pelvis-lumbar mobility is associated with hip function and clinical outcomes in FAIS patients. The HPLMT is an efficient tool for assessing FAIS patients.

In Silico Drug Repurposing by Structural Alteration after Induced Fit: Discovery of a Candidate Agent for Recovery of Nucleotide Excision Repair in Xeroderma Pigmentosum Group D Mutant (R683W).

Xeroderma pigmentosum complementation group D (XPD) is a UV-sensitive syndrome and a rare incurable genetic disease which is caused by the genetic mutation of the excision repair cross-complementation group 2 gene (ERCC2). Patients who harbor only XPD R683W mutant protein develop severe photosensitivity and progressive neurological symptoms. Cultured cells derived from patients with XPD (XPD R683W cells) demonstrate a reduced nucleotide excision repair (NER) ability. We hope to ameliorate clinical symptoms if we can identify candidate agents that would aid recovery of the cells' NER ability. To investigate such candidates, we created in silico methods of drug repurposing (in silico DR), a strategy that utilizes the recovery of ATP-binding in the XPD R683W protein after the induced fit. We chose 4E1RCat and aprepitant as the candidates for our in silico DR, and evaluated them by using the UV-induced unscheduled DNA synthesis (UDS) assay to verify the recovery of NER in XPD R683W cells. UDS values of the cells improved about 1.4-1.7 times after 4E1RCat treatment compared with solvent-only controls; aprepitant showed no positive effect. In this study, therefore, we succeeded in finding the candidate agent 4E1RCat for XPD R683W. We also demonstrated that our in silico DR method is a cost-effective approach for drug candidate discovery.

Rad18 mediates specific mutational signatures and shapes the genomic landscape of carcinogen-induced tumors in vivo.

The E3 ubiquitin ligase Rad18 promotes a damage-tolerant and error-prone mode of DNA replication termed trans-lesion synthesis that is pathologically activated in cancer. However, the impact of vertebrate Rad18 on cancer genomes is not known. To determine how Rad18 affects mutagenesis in vivo, we have developed and implemented a novel computational pipeline to analyze genomes of carcinogen (7, 12-Dimethylbenz[a]anthracene, DMBA)-induced skin tumors from Rad18+/+ and Rad18- / - mice. We show that Rad18 mediates specific mutational signatures characterized by high levels of A(T)>T(A) single nucleotide variations (SNVs). In Rad18- /- tumors, an alternative mutation pattern arises, which is characterized by increased numbers of deletions >4 bp. Comparison with annotated human mutational signatures shows that COSMIC signature 22 predominates in Rad18+/+ tumors whereas Rad18- / - tumors are characterized by increased contribution of COSMIC signature 3 (a hallmark of BRCA-mutant tumors). Analysis of The Cancer Genome Atlas shows that RAD18 expression is strongly associated with high SNV burdens, suggesting RAD18 also promotes mutagenesis in human cancers. Taken together, our results show Rad18 promotes mutagenesis in vivo, modulates DNA repair pathway choice in neoplastic cells, and mediates specific mutational signatures that are present in human tumors.

RAD18 mediates DNA double-strand break-induced ubiquitination of chromatin protein.

The E3 ubiquitin ligase RAD18 mono-ubiquitinates PCNA to promote bypass of replication fork-stalling DNA lesions. On the other hand, RAD18 also contributes to DNA double-strand break (DSB) repair. RAD18 is recruited to ionizing radiation (IR)-induced DSB and colocalizes with ubiquitinated chromatin proteins. RAD18 interacts with the ubiquitinated chromatin proteins via its ubiquitin-binding Zinc finger (UBZ) domain and is proposed to propagate DNA DSB signalling and recruit DNA repair proteins. We found that purified human RAD18 protein complexed with RAD6B (RAD6B-RAD18) catalyzes mono- and poly-ubiquitination of histone H2A in vitro while UBZ domain-mutated RAD18 complexed with RAD6B protein catalyzes mono- but not poly-ubiquitination of histone H2A. Human RAD18-/-cells synchronized at the G1 phase show a reduced signal of ubiquitinated protein in chromatin after IR when compared to that of wild-type control cells. The reduced signal of ubiquitinated protein in RAD18-/-cells is rescued by the introduction of RAD18 cDNA but to a lesser extent by the introduction of cDNA coding RAD18 lacking UBZ domain. Taken together, these results indicate that RAD18 mediates DSB-induced ubiquitination of chromatin protein during the G1 phase.

Roles of Chk2/CHEK2 in guarding against environmentally induced DNA damage and replication-stress.

Checkpoint kinase 2 (human CHEK2; murine Chk2) is a critical mediator of the DNA damage response and has established roles in DNA double strand break (DSB)-induced apoptosis and cell cycle arrest. DSBs may be invoked directly by ionizing radiation but may also arise indirectly from environmental exposures such as solar ultraviolet (UV) radiation. The primary forms of DNA damage induced by UV are DNA photolesions (such as cyclobutane pyrimidine dimers CPD and 6-4 photoproducts) which interfere with DNA synthesis and lead to DNA replication fork stalling. Persistently stalled and unresolved DNA replication forks can "collapse" to generate DSBs that induce signaling via Chk2 and its upstream activator the ataxia telangiectasia-mutated (ATM) protein kinase. This review focuses on recently defined roles of Chk2 in protecting against DNA replication-associated genotoxicity. Several DNA damage response factors such as Rad18, Nbs1 and Chk1 suppress stalling and collapse of DNA replication forks. Defects in the primary responders to DNA replication fork stalling lead to generation of DSB and reveal "back-up" roles for Chk2 in S-phase progression and genomic stability. In humans, there are numerous variants of the CHEK2 gene, including CHEK2*1100delC. Individuals with the CHEK2*1100delC germline alteration have an increased risk of developing breast cancer and malignant melanoma. DNA replication fork-stalling at estrogen-DNA adducts and UV-induced photolesions are implicated in the etiology of breast cancer and melanoma, respectively. It is likely therefore that the Chk2/CHEK2-deficiency is associated with elevated risk for tumorigenesis caused by replication-associated genotoxicities that are exacerbated by environmental genotoxins and intrinsic DNA-damaging agents.

Validity of the Japanese Orthopaedic Association Hip Disease Evaluation Questionnaire (JHEQ) for Japanese patients with labral tear.

The Japanese Orthopaedic Association Hip Disease Evaluation Questionnaire (JHEQ) was created for patient-reported outcome measures (PROMs) and to evaluate the conditions of patients with hip disease. Nevertheless, the validity of the JHEQ for patients with hip labral tears remains unclear. Therefore, we validated the JHEQ in patients with labral tears. There were 51 patients (mean age 44.5, range 18-60 years; 31 women). Thirty-two patients had right-sided tears, 29 underwent hip arthroscopy, 32 had femoroacetabular impingement and 15 had developmental hip dysplasia. Five PROMs included in the JHEQ were evaluated using test-retest methods. Statistical analysis was performed using SPSS software according to the COnsensus-based Standards for the selection of health status Measurement Instruments checklist. The intra-class correlation coefficient (1, 2) of all JHEQ scores (84 points) was 0.88 and Cronbach's α was 0.94. Bland-Altman analysis revealed good test-retest reliability for the JHEQ. The Spearman's rank test, including the SF-36 subscale, showed a high correlation with physical functioning [1, 0.67 (P < 0.01); 2, 0.65 (P < 0.01)], body pain [1, 0.54 (P < 0.01); 2, 0.53 (P < 0.01)] and physical component summary [1, 0.55 (P < 0.01); 2, 0.55 (P < 0.01)]. The value of minimal important change (22.9) was higher than that of smallest detectable change (3.21), suggesting that the JHEQ has adequate responsiveness. We demonstrated the reliability, validity and responsiveness of the JHEQ in Japanese patients with hip labral tears. JHEQ is a valid assessment tool not only for patients with hip osteoarthritis or osteonecrosis but also for those with hip labral tears.

Arthroscopic anterior inferior iliac spine decompression does not alter postoperative muscle strength.

PURPOSE: The purpose of this study was to assess the additional effect of anterior inferior iliac spine (AIIS) decompression on knee extensor and hip flexor strength and compare functional outcomes after arthroscopic FAI correction with and without AIIS decompression. METHODS: Sixty patients who underwent arthroscopic FAI correction surgery were divided into two groups matched for AIIS morphology: 31 patients who underwent arthroscopic FAI surgery only (without AIIS decompression) (FAI group) (AIIS Type I; n = 5, Type II; n = 26, Type III; n = 0) and 29 patients who underwent arthroscopic FAI surgery with AIIS decompression (AIIS group) (AIIS Type I; n = 5, Type II; n = 24, Type III; n = 0). Knee extensor and hip flexor strength were evaluated preoperatively and at 6 months after surgery. Patient-reported outcome (PRO) scores using the modified Harris hip score (MHHS), the nonarthritic hip score (NAHS) and iHOT-12 were obtained preoperatively and at 6 months after surgery. RESULTS: In the AIIS group, there was no significant difference between knee extensor strength pre- and postoperatively (n.s.). In the AIIS group, hip flexor strength was significantly improved postoperatively compared to preoperative measures (p < 0.05). In the FAI group, there were no significant improvements regarding muscle strength (n.s.). While there were no significant differences of preoperative and postoperative MHHS and NAHS between both groups (MHHS; n.s., NAHS; n.s.), the mean postoperative iHOT-12 in the FAI group was inferior to that in the AIIS group. (p < 0.01). The revision surgery rate for the AIIS group was significantly lower compared with that in the FAI group (p < 0.05). CONCLUSION: Anterior inferior iliac spine decompression, as a part of an arthroscopic FAI corrective procedure, had a lower revision surgery rate and did not compromise knee extensor and hip flexor strength, and it improved clinical outcomes comparable to FAI correction without AIIS decompression. AIIS decompression for FAI correction improved postoperative PRO scores without altering the muscle strength of hip flexor and knee extensor. LEVEL OF EVIDENCE: III.

Exploring the validation of a Japanese version of the International Hip Outcome Tool 12: Reliability, validity, and responsiveness.

BACKGROUNDS: The International Hip Outcome Tool 12 (iHOT12) was authorized by the Multicenter Arthroscopy of the Hip Outcomes Research Network (MAHORN). iHOT12 is increasingly being adopted in orthopedic studies to report patient outcomes. This study aimed to develop a Japanese version of the International Hip Outcome Tools "iHOT12J", and to establish its reliability, validity, and responsiveness. METHODS: To assess test-retest reliability, an identical set of patients reported outcome measures with five qualitative scoring measures including iHOT12; these were filled out by each patient twice. Reliability was explored using Cronbachss alpha and intraclass correlation coefficient. The Bland-Altman plot was used to explore the absolute agreement. To evaluate validity, we examined the relationships between SF36 and iHOT12. Responsiveness was assessed by comparing the smallest detectable change to the minimal important change by applying an anchor-based approach. RESULTS: Fifty patients (51 joints) were analyzed from March 2016 to October 2017 in Japanese four facility. The Cronbach α coefficient was 0.90 and the average value of intraclass coefficient (ICC) was 0.89. Bland-Altman plot analysis showed a solid agreement. Regarding the validity, the Spearman rank correlation coefficients were strong with PF (r = 0.69, p < 0.01), BP (r = 0.71, p < 0.01) and PCS (r = 0.69, p < 0.01). The smallest detectable change (3.19) was smaller than the minimum important change (12.40). CONCLUSIONS: We developed iHOT12J, which seems to show sufficient reliability, validity, and responsiveness. We believe that this patient reported outcome measure is beneficial in studying Japanese patients with femoroacetabular impingement.

Japanese version of the international PROMs "Vail Hip Score": Reliability, validity, and responsiveness according to the COSMIN checklist.

BACKGROUND: This study aimed to develop a Japanese version of the international PROMs "Vail Hip Score (Vail10)" and to establish its reliability, validity, and responsiveness with COSMIN check-list. METHODS: The study was conducted from March 2016 to October 2017 and included 46 patients totaling 47 joints. Disorders included 30 cases of FAI (55%), 13 cases of DDH (28%), and 4 others (8%). We administered an identical set of PROMs (5 measures: Japanese-version iHOT12 (pilot draft), Japanese-version Vail10, Japanese-version Oxford Hip Score, JHEQ, and SF36) twice in these subjects. We determined interclass correlation coefficients for the first and second round [ICC(1,2)], as well as the Cronbach α coefficient for patient responses to each of the 10 items in Vail10. In addition, we determined Spearman rank correlation coefficients of Vail10, OHS, JHEQ, satisfaction VAS, the 8 subscales of SF36, and the 3 QOL summary scores. RESULTS: ICC for the total score of all 10 items in Vail10 was 0.96. Cronbach α coefficient was 0.96. Bland-Altman plot analysis showed a solid agreement. Regarding the validity, Spearman rank correlation coefficients, only satisfaction VAS, and SF36 subscales of PF and BP had r > 0.45 (p < 0.01 in both administration rounds). The SDC (1.32) was smaller than the MIC (8.14). CONCLUSIONS: After developing the Japanese version of Vail10, we examined its Reliability, validity, and responsiveness by administering the measure to patients with acetabular labral tear. Correlations were strong and demonstrated the efficacy of the Japanese version of Vail10.

DNA repair protein Rad18 restricts LINE-1 mobility.

Long interspersed element-1 (LINE-1, L1) is a mobile genetic element comprising about 17% of the human genome. L1 utilizes an endonuclease to insert L1 cDNA into the target genomic DNA, which induces double-strand DNA breaks in the human genome and activates the DNA damage signaling pathway, resulting in the recruitment of DNA-repair proteins. This may facilitate or protect L1 integration into the human genome. Therefore, the host DNA repair machinery has pivotal roles in L1 mobility. In this study, we have, for the first time, demonstrated that the DNA repair protein, Rad18, restricts L1 mobility. Notably, overexpression of Rad18 strongly suppressed L1 retrotransposition as well as L1-mediated Alu retrotransposition. In contrast, L1 retrotransposition was enhanced in Rad18-deficient or knockdown cells. Furthermore, the Rad6 (E2 ubiquitin-conjugated enzyme)-binding domain, but not the Polη-binding domain, was required for the inhibition of L1 retrotransposition, suggesting that the E3 ubiquitin ligase activity of Rad18 is important in regulating L1 mobility. Accordingly, wild-type, but not the mutant Rad18-lacking Rad6-binding domain, bound with L1 ORF1p and sequestered with L1 ORF1p into the Rad18-nuclear foci. Altogether, Rad18 restricts L1 and Alu retrotransposition as a guardian of the human genome against endogenous retroelements.

Differential Roles of Rad18 and Chk2 in Genome Maintenance and Skin Carcinogenesis Following UV Exposure.

Defects in DNA polymerase Eta (Polη) cause the sunlight-sensitivity and skin cancer-propensity disorder xeroderma pigmentosum variant. The extent to which Polη function depends on the upstream E3 ubiquitin ligase Rad18 is controversial and has not been investigated using mouse models. Therefore, we tested the role of Rad18 in UV-inducible skin tumorigenesis. Because Rad18 deficiency leads to compensatory DNA damage signaling by Chk2, we also investigated genetic interactions between Rad18 and Chk2 in vivo. Chk2-/-Rad18-/- mice were prone to spontaneous lymphomagenesis. Both Chk2-/- and Chk2-/-Rad18-/- mice were prone to UV-B irradiation-induced skin tumorigenesis when compared with wild-type (WT) animals, but unexpectedly Rad18-/- mice did not recapitulate the skin tumor propensity of Polη mutants. UV-irradiated Rad18-/- cells were more susceptible to G1/S arrest and apoptosis than WT cultures. Chk2 deficiency alleviated both UV-induced G1/S phase arrest and apoptosis of WT and Rad18-/- cells, but led to increased genomic instability. Taken together, our results demonstrate that the tumor-suppressive role of Polη in UV-treated skin is Rad18 independent. We also define a role for Chk2 in suppressing UV-induced skin carcinogenesis in vivo. This study identifies Chk2 dysfunction as a potential risk factor for sunlight-induced skin tumorigenesis in humans.

[Effective Techniques to Reduce Radiation Exposure to Medical Staff during Assist of X-ray Computed Tomography Examination].

Medical staffs like radiological technologists, doctors, and nurses are at an increased risk of exposure to radiation while assisting the patient in a position or monitor contrast medium injection during computed tomography (CT). However, methods to protect medical staff from radiation exposure and protocols for using radiological protection equipment have not been standardized and differ among hospitals. In this study, the distribution of scattered X-rays in a CT room was measured by placing electronic personal dosimeters in locations where medical staff stands beside the CT scanner gantry while assisting the patient and the exposure dose was measured. Moreover, we evaluated non-uniform exposure and revealed effective techniques to reduce the exposure dose to medical staff during CT. The dose of the scattered X-rays was the lowest at the gantry and at the examination table during both head and abdominal CT. The dose was the highest at the trunk of the upper body of the operator corresponding to a height of 130 cm during head CT and at the head corresponding to a height of 150 cm during abdominal CT. The maximum dose to the crystalline lens was approximately 600 µSv during head CT. We found that the use of volumetric CT scanning and X-ray protective goggles, and face direction toward the gantry reduced the exposure dose, particularly to the crystalline lens, for which lower equivalent dose during CT scan has been recently recommended in the International Commission on Radiological Protection Publication 118.

Diverse roles of RAD18 and Y-family DNA polymerases in tumorigenesis.

Mutagenesis is a hallmark and enabling characteristic of cancer cells. The E3 ubiquitin ligase RAD18 and its downstream effectors, the 'Y-family' Trans-Lesion Synthesis (TLS) DNA polymerases, confer DNA damage tolerance at the expense of DNA replication fidelity. Thus, RAD18 and TLS polymerases are attractive candidate mediators of mutagenesis and carcinogenesis. The skin cancer-propensity disorder xeroderma pigmentosum-variant (XPV) is caused by defects in the Y-family DNA polymerase Pol eta (Polη). However it is unknown whether TLS dysfunction contributes more generally to other human cancers. Recent analyses of cancer genomes suggest that TLS polymerases generate many of the mutational signatures present in diverse cancers. Moreover biochemical studies suggest that the TLS pathway is often reprogrammed in cancer cells and that TLS facilitates tolerance of oncogene-induced DNA damage. Here we review recent evidence supporting widespread participation of RAD18 and the Y-family DNA polymerases in the different phases of multi-step carcinogenesis.

DNA repair factor RAD18 and DNA polymerase Polκ confer tolerance of oncogenic DNA replication stress.

The mechanisms by which neoplastic cells tolerate oncogene-induced DNA replication stress are poorly understood. Cyclin-dependent kinase 2 (CDK2) is a major mediator of oncogenic DNA replication stress. In this study, we show that CDK2-inducing stimuli (including Cyclin E overexpression, oncogenic RAS, and WEE1 inhibition) activate the DNA repair protein RAD18. CDK2-induced RAD18 activation required initiation of DNA synthesis and was repressed by p53. RAD18 and its effector, DNA polymerase κ (Polκ), sustained ongoing DNA synthesis in cells harboring elevated CDK2 activity. RAD18-deficient cells aberrantly accumulated single-stranded DNA (ssDNA) after CDK2 activation. In RAD18-depleted cells, the G2/M checkpoint was necessary to prevent mitotic entry with persistent ssDNA. Rad18-/- and Polκ-/- cells were highly sensitive to the WEE1 inhibitor MK-1775 (which simultaneously activates CDK2 and abrogates the G2/M checkpoint). Collectively, our results show that the RAD18-Polκ signaling axis allows tolerance of CDK2-mediated oncogenic stress and may allow neoplastic cells to breach tumorigenic barriers.

Somatic hypermutation of immunoglobulin genes in Rad18 knockout mice.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is triggered by the activity of activation-induced cytidine deaminase (AID). AID induces DNA lesions in variable regions of Ig genes, and error-prone DNA repair mechanisms initiated in response to these lesions introduce the mutations that characterize SHM. Error-prone DNA repair in SHM is proposed to be mediated by low-fidelity DNA polymerases such as those that mediate trans-lesion synthesis (TLS); however, the mechanism by which these enzymes are recruited to AID-induced lesions remains unclear. Proliferating cell nuclear antigen (PCNA), the sliding clamp for multiple DNA polymerases, undergoes Rad6/Rad18-dependent ubiquitination in response to DNA damage. Ubiquitinated PCNA promotes the replacement of the replicative DNA polymerase stalled at the site of a DNA lesion with a TLS polymerase. To examine the potential role of Rad18-dependent PCNA ubiquitination in SHM, we analyzed Ig gene mutations in Rad18 knockout (KO) mice immunized with T cell-dependent antigens. We found that SHM in Rad18 KO mice was similar to wild-type mice, suggesting that Rad18 is dispensable for SHM. However, residual levels of ubiquitinated PCNA were observed in Rad18 KO cells, indicating that Rad18-independent PCNA ubiquitination might play a role in SHM.

A neomorphic cancer cell-specific role of MAGE-A4 in trans-lesion synthesis.

Trans-lesion synthesis (TLS) is an important DNA-damage tolerance mechanism that permits ongoing DNA synthesis in cells harbouring damaged genomes. The E3 ubiquitin ligase RAD18 activates TLS by promoting recruitment of Y-family DNA polymerases to sites of DNA-damage-induced replication fork stalling. Here we identify the cancer/testes antigen melanoma antigen-A4 (MAGE-A4) as a tumour cell-specific RAD18-binding partner and an activator of TLS. MAGE-A4 depletion from MAGE-A4-expressing cancer cells destabilizes RAD18. Conversely, ectopic expression of MAGE-A4 (in cell lines lacking endogenous MAGE-A4) promotes RAD18 stability. DNA-damage-induced mono-ubiquitination of the RAD18 substrate PCNA is attenuated by MAGE-A4 silencing. MAGE-A4-depleted cells fail to resume DNA synthesis normally following ultraviolet irradiation and accumulate γH2AX, thereby recapitulating major hallmarks of TLS deficiency. Taken together, these results demonstrate a mechanism by which reprogramming of ubiquitin signalling in cancer cells can influence DNA damage tolerance and probably contribute to an altered genomic landscape.

Rad18 confers hematopoietic progenitor cell DNA damage tolerance independently of the Fanconi Anemia pathway in vivo.

In cultured cancer cells the E3 ubiquitin ligase Rad18 activates Trans-Lesion Synthesis (TLS) and the Fanconi Anemia (FA) pathway. However, physiological roles of Rad18 in DNA damage tolerance and carcinogenesis are unknown and were investigated here. Primary hematopoietic stem and progenitor cells (HSPC) co-expressed RAD18 and FANCD2 proteins, potentially consistent with a role for Rad18 in FA pathway function during hematopoiesis. However, hematopoietic defects typically associated with fanc-deficiency (decreased HSPC numbers, reduced engraftment potential of HSPC, and Mitomycin C (MMC) -sensitive hematopoiesis), were absent in Rad18(-/-) mice. Moreover, primary Rad18(-/-) mouse embryonic fibroblasts (MEF) retained robust Fancd2 mono-ubiquitination following MMC treatment. Therefore, Rad18 is dispensable for FA pathway activation in untransformed cells and the Rad18 and FA pathways are separable in hematopoietic cells. In contrast with responses to crosslinking agents, Rad18(-/-) HSPC were sensitive to in vivo treatment with the myelosuppressive agent 7,12 Dimethylbenz[a]anthracene (DMBA). Rad18-deficient fibroblasts aberrantly accumulated DNA damage markers after DMBA treatment. Moreover, in vivo DMBA treatment led to increased incidence of B cell malignancy in Rad18(-/-) mice. These results identify novel hematopoietic functions for Rad18 and provide the first demonstration that Rad18 confers DNA damage tolerance and tumor-suppression in a physiological setting.

RAD18 activates the G2/M checkpoint through DNA damage signaling to maintain genome integrity after ionizing radiation exposure.

The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both in vitro and in vivo. Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.