USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.

Mammalian DNA replication employs several RecQ DNA helicases to orchestrate the faithful duplication of genetic information. Helicase function is often coupled to the activity of specific nucleases, but how helicase and nuclease activities are co-directed is unclear. Here we identify the inactive ubiquitin-specific protease, USP50, as a ubiquitin-binding and chromatin-associated protein required for ongoing replication, fork restart, telomere maintenance and cellular survival during replicative stress. USP50 supports WRN:FEN1 at stalled replication forks, suppresses MUS81-dependent fork collapse and restricts double-strand DNA breaks at GC-rich sequences. Surprisingly we find that cells depleted for USP50 and recovering from a replication block exhibit increased DNA2 and RECQL4 foci and that the defects in ongoing replication, poor fork restart and increased fork collapse seen in these cells are mediated by DNA2, RECQL4 and RECQL5. These data define a novel ubiquitin-dependent pathway that promotes the balance of helicase: nuclease use at ongoing and stalled replication forks.

PrimPol-dependent single-stranded gap formation mediates homologous recombination at bulky DNA adducts.

Stalled replication forks can be restarted and repaired by RAD51-mediated homologous recombination (HR), but HR can also perform post-replicative repair after bypass of the obstacle. Bulky DNA adducts are important replication-blocking lesions, but it is unknown whether they activate HR at stalled forks or behind ongoing forks. Using mainly BPDE-DNA adducts as model lesions, we show that HR induced by bulky adducts in mammalian cells predominantly occurs at post-replicative gaps formed by the DNA/RNA primase PrimPol. RAD51 recruitment under these conditions does not result from fork stalling, but rather occurs at gaps formed by PrimPol re-priming and resection by MRE11 and EXO1. In contrast, RAD51 loading at double-strand breaks does not require PrimPol. At bulky adducts, PrimPol promotes sister chromatid exchange and genetic recombination. Our data support that HR at bulky adducts in mammalian cells involves post-replicative gap repair and define a role for PrimPol in HR-mediated DNA damage tolerance.

Isomerization of BRCA1-BARD1 promotes replication fork protection.

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.

MRE11 as a Predictive Biomarker of Outcome After Radiation Therapy in Bladder Cancer.

PURPOSE: Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials. METHODS AND MATERIALS: Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom. RESULTS: Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival. CONCLUSIONS: Manual IHC scoring of MRE11 was not validated as a reproducible biomarker of radiation-based bladder preservation success. There is a need for automated quantitative methods or a reassessment of how DNA-damage response relates to clinical outcomes.

The deSUMOylase SENP2 coordinates homologous recombination and nonhomologous end joining by independent mechanisms.

SUMOylation (small ubiquitin-like modifier) in the DNA double-strand break (DSB) response regulates recruitment, activity, and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and nonhomologous end joining (NHEJ) through the investigation of the deSUMOylase SENP2. We found that regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast, we show that HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 focus retention and increases NHEJ and radioresistance. Collectively, our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

MRE11A Isoform Expression Associated with Outcome Following Radiotherapy in Muscle-Invasive Bladder Cancer does not Alter Cell Survival and DNA Double-Strand Break Repair Following Ionising Radiation.

BACKGROUND: DNA double strand breaks are the cytotoxic lesions produced by ionising radiation. Critical for the repair of these lesions is the DNA damage response protein MRE11 which, in a complex with RAD50 and NBS1, mediates DNA damage signalling and double-strand break repair. We previously found the presence of an MRE11 germline single nucleotide polymorphism (SNP), rs1805363 (G > A), to be associated with poor outcome following radiotherapy (RT) and increased expression of MRE11 isoform 2 in a limited panel of bladder cancer cell lines and tumours. OBJECTIVES: To look for further evidence in support of the SNP/isoform association in a larger panel of germline and tumour samples donated by patients diagnosed with invasive bladder cancer, and to test the hypothesis that bladder cancer cells expressing MRE11 isoform 2 would be more radio resistant than cells expressing MRE11 isoform 1. METHODS: Germline DNA from 189 patients with invasive bladder cancer (141 T2, 48 T1) was genotyped for the rs1805363 G > A SNP. Loss of heterozygosity was determined by genotyping tumour DNA in 17GA germline patients. The Cancer Genome Atlas was mined to correlate presence of the GA germline genotype with MRE11 isoform expression. We used colony formation assays and γH2AX foci kinetics after ionising radiation in RT112 MRE11 knockdown cells expressing ectopic MRE11 isoform 1 or 2. RESULTS: Of the 189 germline DNA samples, 22 contained both the A minor allele and G major allele with the remaining wild type containing only the G major allele. LOH was identified in seven of 17 available tumour samples. Tumour MRE11 isoform 2 expression was found to be significantly higher (p = 0.007) in patients's samples containing the A minor allele compared to those with only the G major allele (n = 23). In the TCGA database we found 16% (66 out of 406) of bladder tumours heterozygous for the SNP and only two homozygous, and a significant relative increase of isoform 2 usage (p = 0.017). We identified no significant difference in radio sensitivity between bladder cancer cells expressing either MRE11 isoform. CONCLUSIONS: In this study the MRE11 isoform 2 was not found to be associated with increased cellular sensitivity to radiation. We conclude that the previously reported association between the germline rs1805363 SNP and poor survival in MIBC patients following RT is unlikely to be related to the DNA damage response function of MRE11 isoform 2.

Harnessing citizen science through mobile phone technology to screen for immunohistochemical biomarkers in bladder cancer.

BACKGROUND: Immunohistochemistry (IHC) is often used in personalisation of cancer treatments. Analysis of large data sets to uncover predictive biomarkers by specialists can be enormously time-consuming. Here we investigated crowdsourcing as a means of reliably analysing immunostained cancer samples to discover biomarkers predictive of cancer survival. METHODS: We crowdsourced the analysis of bladder cancer TMA core samples through the smartphone app 'Reverse the Odds'. Scores from members of the public were pooled and compared to a gold standard set scored by appropriate specialists. We also used crowdsourced scores to assess associations with disease-specific survival. RESULTS: Data were collected over 721 days, with 4,744,339 classifications performed. The average time per classification was approximately 15 s, with approximately 20,000 h total non-gaming time contributed. The correlation between crowdsourced and expert H-scores (staining intensity × proportion) varied from 0.65 to 0.92 across the markers tested, with six of 10 correlation coefficients at least 0.80. At least two markers (MRE11 and CK20) were significantly associated with survival in patients with bladder cancer, and a further three markers showed results warranting expert follow-up. CONCLUSIONS: Crowdsourcing through a smartphone app has the potential to accurately screen IHC data and greatly increase the speed of biomarker discovery.

No additional prognostic value for MRE11 in squamous cell carcinomas of the anus treated with chemo-radiotherapy.

This corrects the article DOI: 10.1038/bjc.2017.188.

No additional prognostic value for MRE11 in squamous cell carcinomas of the anus treated with chemo-radiotherapy.

BACKGROUND: The majority of anal cancers (84-95%) are driven by infection with human papillomavirus (HPV). HPV-positive tumours show significantly better responses to chemo-radiotherapy when compared with HPV-negative tumours. HPV infection is linked to alterations in DNA damage response proteins, including MRE11. MRE11 is a potential predictive biomarker for response to radiotherapy in muscle-invasive bladder cancer and may hold predictive power in other cancers. METHODS: Using a previously reported cohort, we evaluated the levels of MRE11 in anal cancer and assessed its predictive value in this disease. RESULTS: We found no association between the level of MRE11 and relapse-free survival following chemo-radiotherapy. CONCLUSIONS: MRE11 has no predictive value in the analysis of relapse-free survival after chemo-radiotherapy in anal cancer and does not add to the prognostic value of p16 and tumour-infiltrating lymphocyte scores. Further investigation into the role of DNA repair proteins in anal cancer is required.

Impact of PNKP mutations associated with microcephaly, seizures and developmental delay on enzyme activity and DNA strand break repair.

Microcephaly with early-onset, intractable seizures and developmental delay (MCSZ) is a hereditary disease caused by mutations in polynucleotide kinase/phosphatase (PNKP), a DNA strand break repair protein with DNA 5'-kinase and DNA 3'-phosphatase activity. To investigate the molecular basis of this disease, we examined the impact of MCSZ mutations on PNKP activity in vitro and in cells. Three of the four mutations currently associated with MCSZ greatly reduce or ablate DNA kinase activity of recombinant PNKP at 30°C (L176F, T424Gfs48X and exon15Δfs4X), but only one of these mutations reduces DNA phosphatase activity under the same conditions (L176F). The fourth mutation (E326K) has little impact on either DNA kinase or DNA phosphatase activity at 30°C, but is less stable than the wild-type enzyme at physiological temperature. Critically, all of the MCSZ mutations identified to date result in ∼ 10-fold reduced cellular levels of PNKP protein, and reduced rates of chromosomal DNA strand break repair. Together, these data suggest that all four known MCSZ mutations reduce the cellular stability and level of PNKP protein, with three mutations likely ablating cellular DNA 5'-kinase activity and all of the mutations greatly reducing cellular DNA 3'-phosphatase activity.