A Roberts Syndrome Individual With Differential Genotoxin Sensitivity and a DNA Damage Response Defect

Abstract

Syndrome individual with differential genotoxin sensitivity and a DNA damage response defect, SUMMARY Purpose: Roberts Syndrome (RBS) is a rare recessively-transmitted developmental disorder characterized by growth retardation, craniofacial abnormalities and truncation of limbs. All affected individuals to date have mutations in the ESCO2 (Establishment of cohesion 2) gene, a key regulator of the cohesin complex, which is involved in sister chromatid cohesion and DNA double-strand break (dsb) repair.Here we characterize DNA damage responses (DDRs) for the first time in a RBSaffected family. Methods and Materials: Lymphoblastoid cell lines (LCLs) were established from an RBS family, including the proband, and parents carrying ESCO2 mutations. Various DDR assays were performed on these cells, including clonogenic, chromosome break and apoptosis assays, checkpoint activation indicators and measures of DNA breakage and repair. Results: Cells derived from the RBS-affected individual showed sensitivity to ionizing radiation (IR) and Mitomycin C (MMC) -induced DNA damage. In this ESCO2 compound heterozygote, other DNA damage responses were also defective, including enhanced IR-induced clastogenicity and apoptosis, increased DNA dsb induction and a reduced capacity for repairing IR -induced DNA dsbs as measured by γ-H2AX foci and the comet assay.Conclusions: in addition to its developmental features, RBS can be, like ataxia telangiectasia, considered a DNA damage response-defective syndrome, which contributes to its cellular, molecular and clinical phenotype.

DNA damage induces Yap5-dependent transcription of ECO1/CTF7 in Saccharomyces cerevisiae

“…It is important to note in addition that both human and yeast models of RBS exhibit genotoxic sensitivity. We posit that defects in appropriate DNA damage responses, that include a failure to upregulate ESCO2, may constitute a critical but as yet unrecognized role in developmental defects [81][82][83]. The findings presented here demonstrate the complexity of cellular responses to DNA damage and provide tools for future endeavors that may further link Eco1 function and expression in disease and development.…”

An ever-changing landscape in Roberts syndrome biology: Implications for macromolecular damage

“…A link between RBS and DNA damage repair-deficient syndromes is evident from numerous studies. RBS patient cells exhibit hypersensitivities to a broad range of genotoxic agents that include the DNA cross-linker mitomycin C (MMC), ionizing radiation (IR), the topoisomerase II inhibitors etoposide, and the topoisomerase I inhibitor camptothecin (CPT) [ 5 , 10 , 64 , 71 – 74 ]. The role for cohesin in DNA damage responses includes both serving as a direct phosphorylation target of checkpoint kinases and promoting DNA repair [ 30 , 31 , 75 – 80 ].…”

“…This raises the possibility that RBS cell sensitivity to genotoxic stress could be attributed to either a failure to respond to DNA damage and/or a failure to repair the damaged DNA. On the one hand, RBS cell lines exhibit phosphorylation of ATM, checkpoint kinase 1 homolog (Chk1), and the tumor suppressor protein (p53) in response to DNA damaging agents [ 64 , 74 ], suggesting that these checkpoints are functional in RBS cells. On the other hand, several lines of evidence suggest that RBS cells exhibit a reduced ability to repair DNA damage after checkpoint activation.…”

“…A combination of western blot analyses of whole cell extracts from RBS patient-derived fibroblasts and “Comet” assays shows increased phosphorylated histone 2A variant X (γ-H2AX) levels and chromosomal fragmentation, respectively, that persist long after IR treatment. These results indicate that double-strand breaks (DSBs) persist into subsequent cell cycles due to inefficient repair [ 74 ], which likely contributes to increased apoptosis levels. Similarly, immortalized RBS fibroblasts, 24 h after IR or MMC exposure, contain a decreased number of repair recombinase Rad51 foci, relative to control cells [ 64 ].…”

“…Is it possible that ESCO2 mutation is, in itself, sufficient to produce ROS? In fact, human RBS models and yeast cohesion mutants exhibit markers of increased ROS levels both in the absence of challenges and in response to DNA damage [ 63 , 74 , 97 , 99 , 100 ]. Mutual causality between DNA damage and ROS production may provide a synergistic mechanism, which enhances mutation rates in RBS cells.…”