Germline Mutation in MUS81 Resulting in Impaired Protein Stability is Associated with Familial Breast and Thyroid Cancer.
ABSTRACT: Multiple primary thyroid cancer (TC) and breast cancer (BC) are commonly diagnosed, and the lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC. Although this phenotype is partially explained by TP53 or PTEN mutations, a significant number of patients are negative for these alterations. We judiciously recruited patients diagnosed with BC and/or TC having a family history of these tumors and assessed their whole-exome sequencing. After variant prioritization, we selected MUS81 c.1292G>A (p.R431H) for further investigation. This variant was genotyped in a healthy population and sporadic BC/TC tissues and investigated at the protein level and cellular models. MUS81 c.1292G>A was the most frequent variant (25%) and the strongest candidate due to its function of double-strand break repair. This variant was confirmed in four relatives from two families. MUS81 p.R431H protein exhibited lower expression levels in tumors from patients positive for the germline variant, compared with wild-type BC, and normal breast and thyroid tissues. Using cell line models, we showed that c.1292G>A induced protein instability and affected DNA damage response. We suggest that MUS81 is a novel candidate involved in familial BC/TC based on its low frequency in healthy individuals and proven effect in protein stability.
Project description:Previous studies have documented an intrinsic association between breast cancer (BC) and thyroid cancer (TC), but the clinical relevance of this relationship is not well defined. In the present study, we specifically investigated the impact of a history of TC on clinical outcomes of BC. We performed a population-based comparative analysis of tumor behaviors and BC-specific mortalities in 427,893 female patients with BC in the USA Surveillance, Epidemiology and End Results 9 database (1973-2013). In this cohort of subjects, 2,569 patients also had a history of differentiated TC (BC/TC), including BC diagnosed before TC (BC-1st) and BC diagnosed after TC (TC-1st), with the median follow-up time of 81 (IQR, 33-160) months. We found that, compared with matched BC-only patients, less aggressive BC tumor behaviors occurred in BC/TC patients, as exemplified by a distant metastasis rate of 7.0% in the former versus 3.3% in the latter (P<0.001). In BC/TC, BC-1st, and TC-1st patients versus their matched BC-only patients, BC-specific mortalities were 11.3% versus 21.0%, 9.9% versus 26.4%, and 12.4% versus 16.9%. These corresponded to hazard ratios (HR) (95% CI) of 0.47 (0.42-0.53), 0.31 (0.26-0.37), and 0.72 (0.61-0.84), respectively (all P<0.001), being lowest in BC-1st patients <50 years old [HR = 0.22 (0.16-0.31)], which remained significant after adjustment for clinicopathological and socioeconomic factors. Estrogen/progesterone receptor expression in BC tumors was significantly higher in patients with BC/TC than matched BC-only patients, providing evidence that BC in the former was biologically unique. Thus, a history of TC, particularly in younger BC-1st patients, may identify BC as a unique disease entity characterized by a decreased disease-specific mortality risk. The results have potentially important clinical and biological implications for BC in this special patient population and encourage further studies to confirm.
Project description:BACKGROUND:Thyroid cancer (TC) is one of the most commonly seen secondary malignancy in breast cancer (BC) survivors. MATERIALS AND METHODS:A retrospective study was conducted in BC patients in our center from 1999 to 2013. Patients were divided into BC-TC group and BC-alone group. RESULTS:In total, 13 978 BC patients were identified, among whom 247 (1.8%) had TC. The standardized incidence ratio (SIR) of TC was 4.48 compared with Chinese females, and up to 98.0% of cases were thyroid papillary carcinomas. A family history of malignancy was the only independent risk factor (odds ratio = 1.457, P = 0.025) for development of TC in patients with BC. We also identified inferior survival in patients with synchronous versus metachronous BC-TC (P = 0.016). Synchronous BC-TC (risk ratio = 5.597, P = 0.018) was an independent prognostic factor for inferior RFS. CONCLUSIONS:We observed high co-occurrence of TC in patients with BC. There might be different mechanisms behind synchronous and metachronous BC-TC.
Project description:Replication forks frequently stall at regions of the genome that are difficult to replicate or contain lesions that cause replication blockage. An important mechanism for the restart of a stalled fork involves endonucleolytic cleavage that can lead to fork restoration and replication progression. Here, we show that the structure-selective endonuclease MUS81-EME2 is responsible for fork cleavage and restart in human cells. The MUS81-EME2 protein, whose actions are restricted to S phase, is also responsible for telomere maintenance in telomerase-negative ALT (Alternative Lengthening of Telomeres) cells. In contrast, the G2/M functions of MUS81, such as the cleavage of recombination intermediates and fragile site expression, are promoted by MUS81-EME1. These results define distinct and temporal roles for MUS81-EME1 and MUS81-EME2 in the maintenance of genome stability.
Project description:Rad54, a key protein of homologous recombination, physically interacts with a DNA structure-specific endonuclease, Mus81-Eme1. Genetic data indicate that Mus81-Eme1 and Rad54 might function together in the repair of damaged DNA. In vitro, Rad54 promotes branch migration of Holliday junctions, whereas the Mus81-Eme1 complex resolves DNA junctions by endonucleolytic cleavage. Here, we show that human Rad54 stimulates Mus81-Eme1 endonuclease activity on various Holliday junction-like intermediates. This stimulation is the product of specific interactions between the human Rad54 (hRad54) and Mus81 proteins, considering that Saccharomyces cerevisiae Rad54 protein does not stimulate human Mus81-Eme1 endonuclease activity. Stimulation of Mus81-Eme1 cleavage activity depends on formation of specific Rad54 complexes on DNA substrates occurring in the presence of ATP and, to a smaller extent, of other nucleotide cofactors. Thus, our results demonstrate a functional link between the branch migration activity of hRad54 and the structure-specific endonuclease activity of hMus81-Eme1, suggesting that the Rad54 and Mus81-Eme1 proteins may cooperate in the processing of Holliday junction-like intermediates during homologous recombination or DNA repair.
Project description:The MUS81-EME1 endonuclease maintains metazoan genomic integrity by cleaving branched DNA structures that arise during the resolution of recombination intermediates. In humans, MUS81 also forms a poorly characterized complex with EME2. Here, we identify and determine the structure of a winged helix (WH) domain from human MUS81, which binds DNA. WH domain mutations greatly reduce binding of the isolated domain to DNA and impact on incision activity of MUS81-EME1/EME2 complexes. Deletion of the WH domain reduces the endonuclease activity of both MUS81-EME1 and MUS81-EME2 complexes, and incisions made by MUS81-EME2 are made closer to the junction on substrates containing a downstream duplex, such as fork structures and nicked Holliday junctions. WH domain mutation or deletion in Schizosaccharomyces pombe phenocopies the DNA-damage sensitivity of strains deleted for mus81. Our results indicate an important role for the WH domain in both yeast and human MUS81 complexes.
Project description:Breast cancer (BC) and thyroid cancer (TC) are common malignancies among females. However, the connection between TC and BC is not well understood. To explore the relationship between these two cancers and to determine the effect of second metachronous TC on BC survival, we compared BC patients with or without second primary TC using data from the Surveillance, Epidemiology, and End Results (SEER) database. We extracted data from patients with only BC or TC and from BC patients with a second metachronous cancer from 2000-2014. Differences in the clinicopathological and treatment characteristics between BC patients with or without second metachronous TC were analyzed by chi-square tests. Multivariate analyses of BC survival were performed by using Cox regression models. Comparison of disease-specific survival (DSS) curves between these cohorts was performed with the log-rank (Mantel-Cox) test. Survival analyses were also performed using data from 1980-1994. Within this dataset, we found 1,262 BC cases in which a second metachronous TC (BC2TC) developed, accounting for 3.1% of all metachronous cancers following BC from 2000-2014. No significant differences were found in molecular markers. In addition, the mean age at BC diagnosis was younger in the BC2TC group than in the BC group (55.418 y vs 60.273 y). Half of the BC2TC patients developed TC in the first three years following BC diagnosis. Patients with BC2TC showed better DSS than those with BC alone from 2000-2014 (P<0.001). However, this superiority was not significant from 1980-1994 (P = 0.579) or for TNM stage I BC (P = 0.927) and grade I BC (P = 0.431) from 2000-2014. In conclusion, the incidence of BC2TC has increased dramatically during the past 15 years. In addition, patients with BC2TC showed better DSS than patients with BC alone, especially in cases from 2000-2014.
Project description:MUS81 plays important cellular roles in the restart of stalled replication forks, the resolution of recombination intermediates and in telomere length maintenance. Although the actions of MUS81-EME1 have been extensively investigated, MUS81 is the catalytic subunit of two human structure-selective endonucleases, MUS81-EME1 and MUS81-EME2. Little is presently known about the activities of MUS81-EME2. Here, we have purified MUS81-EME2 and compared its activities with MUS81-EME1. We find that MUS81-EME2 is a more active endonuclease than MUS81-EME1 and exhibits broader substrate specificity. Like MUS81-EME1, MUS81-EME2 cleaves 3'-flaps, replication forks and nicked Holliday junctions, and exhibits limited endonuclease activity with intact Holliday junctions. In contrast to MUS81-EME1, however, MUS81-EME2 cuts D-loop recombination intermediates and in so doing disengages the D-loop structure by cleaving the 3'-invading strand. Additionally, MUS81-EME2 acts on 5'-flap structures to cleave off a duplex arm, in reactions that cannot be promoted by MUS81-EME1. These studies suggest that MUS81-EME1 and MUS81-EME2 exhibit similar and yet distinct DNA structure selectivity, indicating that the two MUS81 complexes may promote different nucleolytic cleavage reactions in vivo.
Project description:MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3' side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5' side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.
Project description:Telomerase-negative cancer cells maintain their telomeres through the alternative lengthening of telomeres (ALT) pathway. Although a growing body of evidence demonstrates that the ALT mechanism is a post-replicative telomere recombination process, molecular details of this pathway are largely unknown. Here we demonstrate that MUS81, a DNA structure specific recombination endonuclease, has a key role in the maintenance of telomeres in human ALT cells. We find that MUS81 specifically localizes to ALT-associated promyelocytic leukaemia (PML) nuclear bodies (APBs) and associates with telomeric DNA in ALT cells, which is enriched during the G2 phase of the cell cycle. Depletion of MUS81 results in the reduction of ALT-specific telomere recombination and leads to proliferation arrest of ALT cells. In addition, the endonuclease activity of MUS81 is required for recombination-based ALT cell survival, and the interaction of MUS81 with the telomeric repeat-binding factor TRF2 regulates this enzymatic activity, thereby maintaining telomere recombination. Thus, our results suggest that MUS81 is involved in the maintenance of ALT cell survival at least in part by homologous recombination of telomeres.
Project description:The MUS81 protein belongs to a conserved family of DNA structure-specific nucleases that play important roles in DNA replication and repair. Inactivation of the Mus81 gene in mice has no major deleterious consequences for embryonic development, although cancer susceptibility has been reported. We have investigated the role of MUS81 in human cells by acutely depleting the protein using shRNAs. We found that MUS81 depletion from human fibroblasts leads to accumulation of ssDNA and a constitutive DNA damage response that ultimately activates cellular senescence. Moreover, we show that MUS81 is required for efficient replication fork progression during an unperturbed S-phase, and for recovery of productive replication following replication stalling. These results demonstrate essential roles for the MUS81 nuclease in maintenance of replication fork integrity.