Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5' to the lesion by ERCC1-XPF and 3' to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5' incision by ERCC1-XPF precedes the 3' incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a 'cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.

Nucleotide excision repair (NER) is a DNA repair pathway that is responsible for removing a variety of lesions caused by harmful UV light, chemical carcinogens, and environmental mutagens from DNA. NER involves the concerted action of over 30 proteins that sequentially recognize a lesion, excise it in the form of an oligonucleotide, and fill in the resulting gap by repair synthesis. ERCC1-XPF and XPG are structure-specific endonucleases responsible for carrying out the incisions 5′ and 3′ to the damage respectively, culminating in the release of the damaged oligonucleotide. This review focuses on the recent work that led to a greater understanding of how the activities of ERCC1-XPF and XPG are regulated in NER to prevent unwanted cuts in DNA or the persistence of gaps after incision that could result in harmful, cytotoxic DNA structures.

Nucleotide excision repair (NER) removes lesions caused by environmental mutagens or UV light from DNA. A hallmark of NER is the extraordinarily wide substrate specificity, raising the question of how one set of proteins is able to recognize structurally diverse lesions. Two key features of good NER substrates are that they are bulky and thermodynamically destabilize DNA duplexes. To understand what the limiting step in damage recognition in NER is, we set out to test the hypothesis that there is a correlation of the degree of thermodynamic destabilization induced by a lesion, binding affinity to the damage recognition protein XPC-RAD23B and overall NER efficiency. We chose to use acetylaminofluorene (AAF) and aminofluorene (AF) adducts at the C8 position of guanine in different positions within the NarI (GGCGCC) sequence, as it is known that the structures of the duplexes depend on the position of the lesion in this context. We found that the efficiency of NER and the binding affinity of the damage recognition factor XPC-RAD23B correlated with the thermodynamic destabilization induced by the lesion. Our study is the first systematic analysis correlating these three parameters and supports the idea that initial damage recognition by XPC-RAD23B is a key rate-limiting step in NER.

INTRODUCTION: Ten to 18% of ovarian cancer (OC) can be attributed to a hereditary predisposition. The frequency of pathogenic variants (PVs) among women with epithelial ovarian cancer (EOC) diagnosed at an early age has not been well-described. We present the yield of PVs in women with EOC diagnosed ≤30 years (y) who have undergone genetic testing at a commercial laboratory. METHODS: Results for women with OC submitted for multi-gene hereditary cancer panel testing of at least ATM, BRCA1/2, BRIP1, CHEK2, EPCAM, MLH1, MSH2, MSH6, PALB2, PMS2, RAD51C, and RAD51D were reviewed. Yields were compared using two-tailed Fisher exact test. RESULTS: One hundred fifty-three women with OC were diagnosed ≤30y, and 65 (42%) had EOC. Family history of OC was reported for 35 women (26.9%). Five PVs, in BRIP1, CHEK2 (3) and PALB2, were identified in the women with EOC for a positive yield of 7.7%, which is similar to the yield in all women with OC diagnosed ≤30y regardless of histopathologic subtype (5.9%, 9/153, P=.77; ATM, BRIP1, CHEK2 (6), PALB2). The yield of PVs in women with OC diagnosed >30y was significantly higher than either yield in the early-onset group (14.7%, 546/3,704, P<.0013). CONCLUSION: Women with early-onset OC were less likely to have a PV than those diagnosed >30y, though the detected yield was still appreciable. No PVs were identified in BRCA1/2 or the Lynch syndrome genes, highlighting the utility of panel testing and including genes beyond BRCA1/2 for women with OC regardless of age and histopathology.

INTRODUCTION: Collection and utilization of family history is necessary for hereditary cancer risk assessment but standardization is difficult in healthcare practices. The web-based, patient-entered MyGeneticsTree (MGT) can be administered outside of medical encounters, targeting follow-up on high-risk patients. METHODS: MGT was used in women's health offices between October 2015 and July 2018. Patients (users) were invited by providers directly, or found MGT through other means. MGT was accessed on personal computers, mobile devices, or in-office tablets. RESULTS: A total of 1,922 patients completed MGT; the overall completion rate was 85%. The average age was 45 (between 18 and 86) and 93% were female. The majority (87%) of patients were unaffected themselves, but reported a family history of cancer and/or genetic testing. Half (49%) of all patients met clinical guidelines for genetic testing in one of 5 hereditary cancer syndromes. Cancer family history was reported more in maternal (41%) than paternal (29%) relatives, and in first-degree compared to second- or third-degree relatives (85% to 13% or 2%, respectively). Excluding those with a known familial variant, 19.5% (375/1922) pursued multigene hereditary genetic testing, of whom 9% (n=34) were positive for a pathogenic variant. CONCLUSION: These results point to successful adoption of an online, patient-driven hereditary cancer risk assessment identifying patients appropriate for genetic testing. When administered to a patient population outside or within the office, MyGeneticsTree identified unaffected individuals with significant cancer family history. Planned enhancements include continued algorithm updates, optimized patient ease of use, and improved testing uptake for at-risk patients.