Functional analysis of germline RAD51C missense variants highlight the role of RAD51C in replication fork protection.

Monoallelic or biallelic RAD51C germline mutations results in chromosome instability disorders such as Fanconi anemia and cancers. The bona fide function of RAD51C is to assist RAD51 nucleoprotein filament onto single-strand DNA to complete homologous recombination (HR) repair. In addition to HR repair, the role of RAD51C in DNA replication is emerging when replication forks are transiently or irreversibly stalled. We identified novel RAD51C variants of uncertain significance (VUS) from breast, ovarian, pancreatic and gastric cancer patients and functionally characterized the effect of these variants in replication fork protection and double-strand breaks (DSB's) repair. In RAD51C-deficient Chinese hamster CL-V4B cells, expression of RAD51C F164S, A87E, L134S and E49K variants heightened sensitivity to mitomycin C (MMC), etoposide and PARP inhibition. Differently, expression of subset of RAD51C variants R24L, R24W and R212H displayed mild sensitivity to MMC, etoposide and PARP inhibition. Further functional characterization of a subset of variants revealed that Rad51C F164S, A87E, L134S and E49K variants displayed reduced RAD51 foci formation and increased overall nuclear single strand DNA levels in the presence of replication stress. Additionally, DNA fiber assay revealed that RAD51C F164S, A87E, L134S and E49K variants displayed defective replication fork protection upon prolonged fork stalling. Investigations using patient-derived lymphoblastoid cell line carrying heterozygous RAD51C L134S variant showed an impairment in RAD51 chromatin association and replication fork protection, suggestive of deleteriousness of this VUS variant. Overall, our findings provide more insights into molecular roles of RAD51C in replication fork integrity maintenance and in DSB repair.

Automatic DNA replication tract measurement to assess replication and repair dynamics at the single-molecule level.

MOTIVATION: DNA fibre assay has a potential application in genomic medicine, cancer and stem cell research at the single-molecule level. A major challenge for the clinical and research implementation of DNA fibre assays is the slow speed in which manual analysis takes place as it limits the clinical actionability. While automatic detection of DNA fibres speeds up this process considerably, current publicly available software have limited features in terms of their user interface for manual correction of results, which in turn limit their accuracy and ability to account for atypical structures that may be important in diagnosis or investigative studies. We recognize that core improvements can be made to the GUI to allow for direct interaction with automatic results to preserve accuracy as well as enhance the versatility of automatic DNA fibre detection for use in variety of situations. RESULTS: To address the unmet needs of diverse DNA fibre analysis investigations, we propose DNA Stranding, an open-source software that is able to perform accurate fibre length quantification (13.22% mean relative error) and fibre pattern recognition (R > 0.93) with up to six fibre patterns supported. With the graphical interface, we developed, user can conduct semi-automatic analyses which benefits from the advantages of both automatic and manual processes to improve workflow efficiency without compromising accuracy. AVAILABILITY AND IMPLEMENTATION: The software package is available at https://github.com/lgole/DNAStranding. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The H. pylori CagA Oncoprotein Induces DNA Double Strand Breaks through Fanconi Anemia Pathway Downregulation and Replication Fork Collapse.

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

Molecular connections between circadian rhythm and genome maintenance pathways.

Co-ordinated oscillation of mammalian circadian clock and cell cycle is essential for cellular and organismal homeostasis. Existing preclinical, epidemiological, molecular and biochemical evidence reveals a robust interplay between circadian clock, genome instability and cancer. Furthermore, recent investigations have demonstrated that the alterations in circadian clock perturb genome stability by modulating the cell-cycle timing, altering DNA replication fork progression, influencing DNA damage response (DDR) and DNA repair efficiency. In this review, we examine the most recent findings from different eukaryotic model systems and discuss the functional interaction between circadian factors with key DNA replication, DDR and DNA repair genes.

Investigation into the origins of an ancient BRCA1 founder mutation identified among Chinese families in Singapore.

Identification of ancestry-specific pathogenic variants is imperative for diagnostic, treatment, management and prevention strategies, and to understand penetrance/modifiers on risk. Our study aimed to determine the clinical significance of a recurrent BRCA1 c.442-22_442-13del variant of unknown significance identified among 13 carriers from six Chinese families, all with a significant history of breast and/or ovarian cancer. We further aimed to establish whether this was due to a founder effect and explore its origins. Haplotype analysis, using nine microsatellite markers encompassing 2.5 megabase pairs around the BRCA1 locus, identified a common haploblock specific to the variant carriers, confirming a founder effect. Variant age was estimated to date back 77.9 generations to 69 bc using the Gamma approach. On principal component analysis using single nucleotide polymorphisms merged with 1000 Genomes dataset, variant carriers were observed to overlap predominantly with the southern Han Chinese population. To determine pathogenicity of the variant, we assessed the functional effect on RAD51 foci formation as well as replication fork stability upon induction of DNA damage and observed an impaired DNA repair response associated with the variant. In summary, we identified an ancient Chinese founder mutation dating back 77.9 generations, possibly common among individuals of southern Han Chinese descent. Using evidence from phenotypic/family history studies, segregation analysis and functional characterization, the BRCA1 variant was reclassified from uncertain significance to pathogenic.

Multiple neoplasia in a patient with Gitelman syndrome harboring germline monoallelic MUTYH mutation.

Gitelman syndrome is a rare, recessively inherited disease characterized by chronic hypokalemia and hypomagnesemia as a result of defective electrolyte co-transport at the level of the distal convoluted tubule of the kidney. Here, we present the first report of a patient with Gitelman syndrome who developed multiple neoplasia including colorectal polyposis, synchronous colorectal cancers, recurrent breast fibroadenomata and a desmoid tumor. Whole-exome sequencing confirmed germline compound heterozygous mutations of c.179C > T and c.1326C > G in SLC12A3, and in addition, identified a monoallelic germline c.934-2A > G splice site mutation in MUTYH. In vitro, magnesium deficiency potentiated oxidative DNA damage in lymphoblastoid cell lines derived from the same patient. We postulate that monoallelic MUTYH mutations may manifest in the presence of cooperative non-genetic mechanisms, in this case possibly magnesium deficiency from Gitelman syndrome.

Emerging functions of Fanconi anemia genes in replication fork protection pathways.

Germline mutations in Fanconi anemia (FA) genes predispose to chromosome instability syndromes, such as FA and cancers. FA gene products have traditionally been studied for their role in interstrand cross link (ICL) repair. A fraction of FA gene products are classical homologous recombination (HR) factors that are involved in repairing DNA double-strand breaks (DSBs) in an error-free manner. Emerging evidence suggests that, independent of ICL and HR repair, FA genes protect DNA replication forks in the presence of replication stress. Therefore, understanding the precise function of FA genes and their role in promoting genome stability in response to DNA replication stress is crucial for diagnosing FA and FA-associated cancers. Moreover, molecular understanding of the FA pathway will greatly help to establish proper functional assays for variants of unknown significance (VUS), often encountered in clinics. In this short review, we discuss the recently uncovered molecular details of FA genes in replication fork protection pathways. Finally, we examine how novel FA variants predispose to FA and cancer, due to defective replication fork protection activity.

Missense PALB2 germline variant disrupts nuclear localization of PALB2 in a patient with breast cancer.

The PALB2 protein is essential to RAD51-mediated homologous recombination (HR) repair. Germline monoallelic PALB2 pathogenic variants confer significant risks for breast cancer. However, the majority of PALB2 variants remain classified as variants of unknown significance (VUS). We aim to functionally and mechanistically evaluate three novel PALB2 VUS. Patient-derived lymphoblastoid cell lines containing the VUS were analyzed for nuclear localization and foci formation of RAD51 as a measure of HR efficiency. To understand the mechanism underlying the HR deficiency, PALB2 nuclear localization was assessed using immunofluorescence studies. Among these VUS, c.3251C>T (p.Ser1084Leu) occurred in a patient with metastatic breast cancer while c.1054G>C (p.Glu352Gln) and c.1057A>G (p.Lys353Glu) were seen in patients with squamous cell carcinoma of skin and renal cell carcinoma respectively. Variant c.3251C>T was located within the WD40 domain which normally masked the nuclear export signal sequence responsible for nuclear delocalization of PALB2. Correspondingly, c.3251C>T displayed aberrant cytoplasmic localization of PALB2 which led to an impaired RAD51 nuclear localization and foci formation. On the other hand, both c.1054G>C and c.1057A>G showed intact HR functions and nuclear localization of PALB2, consistent with their locations within domains of no known function. Additionally, the prevalence of c.1054G>C was similar among healthy controls and patients with breast cancer (as seen in other studies), suggestive of its non-pathogenicity. In conclusion, our studies provided the functional evidence showing the deleterious effect of c.3251C>T, and non-deleterious effects of c.1054G>C and c.1057A>G. Using the ClinGen Pathogenicity calculator, c.3251C>T remains a VUS while c.1054G>C and c.1057A>G may be classified as likely benign variants.

Functional relationship between p53 and RUNX proteins.

RUNX genes belong to a three-membered family of transcription factors, which are well established as master regulators of development. Of them, aberrations in RUNX3 expression are frequently observed in human malignancies primarily due to epigenetic silencing, which is often overlooked. At the G1 phase of the cell cycle, RUNX3 regulates the restriction (R)-point, a mechanism that decides cell cycle entry. Deregulation at the R-point or loss of RUNX3 results in premature entry into S phase, leading to a proliferative advantage. Inactivation of Runx1 and Runx2 induce immortalization of mouse embryo fibroblast. As a consequence, RUNX loss induces pre-cancerous lesions independent of oncogene activation. p53 is the most extensively studied tumour suppressor. p53 plays an important role to prevent tumour progression but not tumour initiation. Therefore, upon oncogene activation, early inactivation of RUNX genes and subsequent mutation of p53 appear to result in tumour initiation and progression. Recently, transcription-independent DNA repairing roles of RUNX3 and p53 are emerging. Being evolutionarily old genes, it appears that the primordial function of p53 is to protect genome integrity, a function that likely extends to the RUNX gene as well. In this review, we examine the mechanism and sequence of actions of these tumour suppressors in detail.

RUNX Poly(ADP-Ribosyl)ation and BLM Interaction Facilitate the Fanconi Anemia Pathway of DNA Repair.

The Fanconi anemia (FA) pathway is a pivotal genome maintenance network that orchestrates the repair of DNA interstrand crosslinks (ICLs). The tumor suppressors RUNX1 and RUNX3 were shown to regulate the FA pathway independent of their canonical transcription activities, by controlling the DNA damage-dependent chromatin association of FANCD2. Here, in further biochemical characterization, we demonstrate that RUNX3 is modified by PARP-dependent poly(ADP-ribosyl)ation (PARylation), which in turn allows RUNX binding to DNA repair structures lacking transcription-related RUNX consensus motifs. SILAC-based mass spectrometric analysis revealed significant association of RUNX3 with core DNA repair complexes, including PARP1, even in unstressed cells. After DNA damage, the increased interaction between RUNX3 and BLM facilitates efficient FANCD2 chromatin localization. RUNX-Walker motif mutations from breast cancers are impaired for DNA damage-inducible PARylation, unveiling a potential mechanism for FA pathway inactivation in cancers. Our results reinforce the emerging paradigm that RUNX proteins are tumor suppressors with genome gatekeeper function.

Smarcal1-Mediated Fork Reversal Triggers Mre11-Dependent Degradation of Nascent DNA in the Absence of Brca2 and Stable Rad51 Nucleofilaments.

Brca2 deficiency causes Mre11-dependent degradation of nascent DNA at stalled forks, leading to cell lethality. To understand the molecular mechanisms underlying this process, we isolated Xenopus laevis Brca2. We demonstrated that Brca2 protein prevents single-stranded DNA gap accumulation at replication fork junctions and behind them by promoting Rad51 binding to replicating DNA. Without Brca2, forks with persistent gaps are converted by Smarcal1 into reversed forks, triggering extensive Mre11-dependent nascent DNA degradation. Stable Rad51 nucleofilaments, but not RPA or Rad51T131P mutant proteins, directly prevent Mre11-dependent DNA degradation. Mre11 inhibition instead promotes reversed fork accumulation in the absence of Brca2. Rad51 directly interacts with the Pol α N-terminal domain, promoting Pol α and δ binding to stalled replication forks. This interaction likely promotes replication fork restart and gap avoidance. These results indicate that Brca2 and Rad51 prevent formation of abnormal DNA replication intermediates, whose processing by Smarcal1 and Mre11 predisposes to genome instability.

Moonlighting at replication forks - a new life for homologous recombination proteins BRCA1, BRCA2 and RAD51.

Coordination between DNA replication and DNA repair ensures maintenance of genome integrity, which is lost in cancer cells. Emerging evidence has linked homologous recombination (HR) proteins RAD51, BRCA1 and BRCA2 to the stability of nascent DNA. This function appears to be distinct from double-strand break (DSB) repair and is in part due to the prevention of MRE11-mediated degradation of nascent DNA at stalled forks. The role of RAD51 in fork protection resembles the activity described for its prokaryotic orthologue RecA, which prevents nuclease-mediated degradation of DNA and promotes replication fork restart in cells challenged by DNA-damaging agents. Here, we examine the mechanistic aspects of HR-mediated fork protection, addressing the crosstalk between HR and replication proteins.