Homologous Recombination Research Articles

Multiple DNA repair pathways prevent acetaldehyde-induced mutagenesis in yeast.

Acetaldehyde is the primary metabolite of alcohol and is present in many environmental sources including tobacco smoke. Acetaldehyde is genotoxic, whereby it can form DNA adducts and lead to mutagenesis. Individuals with defects in acetaldehyde clearance pathways have increased susceptibility to alcohol-associated cancers. Moreover, a mutation signature specific to acetaldehyde exposure is widespread in alcohol and smoking-associated cancers. However, the pathways that repair acetaldehyde-induced DNA damage and thus prevent mutagenesis are vaguely understood. Here, we used Saccharomyces cerevisiae to delete genes in each of the major DNA repair pathways to identify those that alter acetaldehyde-induced mutagenesis. We observed that loss of functional nucleotide excision repair (NER) had the largest effect on acetaldehyde mutagenesis. In addition, base excision repair (BER), as well as DNA protein crosslink (DPC) repair pathways were involved in modulating acetaldehyde mutagenesis, while mismatch repair (MMR), homologous recombination (HR) and post replication repair are dispensable for acetaldehyde mutagenesis. Acetaldehyde-induced mutations in an NER-deficient (Δrad1) background were dependent on translesion synthesis as well as DNA inter-strand crosslink (ICL) repair. Moreover, whole genome sequencing of the mutated isolates demonstrated an increase in C→A changes coupled with an enrichment of gCn→A changes which is diagnostic of acetaldehyde exposure in yeast and in human cancers. Finally, downregulation of the leading strand replicative polymerase Pol epsilon, but not the lagging strand polymerase, resulted in increased acetaldehyde mutagenesis, indicating that lesions are likely formed on the leading strand. Our findings demonstrate that multiple DNA repair pathways coordinate to prevent acetaldehyde-induced mutagenesis.

RRM1 promotes homologous recombination and radio/chemo-sensitivity via enhancing USP11 and E2F1-mediated RAD51AP1 transcription

Ribonucleotide reductase M1 (RRM1), the catalytic subunit of ribonucleotide reductase, plays a pivotal role in converting ribonucleotides (NTP) into deoxyribonucleotides (dNTP), essential for DNA replication and repair. Elevated RRM1 expression is associated with various human cancers, correlating with poorer prognosis and reduced overall survival rates. Our previous study found that RRM1 will enter the nucleus to promote DNA damage repair. However, the underlying mechanism remains elusive. Here, we unveil a novel role of RRM1 in promoting homologous recombination (HR) by upregulating the expression of RAD51AP1, a critical HR factor, in an E2F1-dependent manner. We demonstrate that RRM1 interacts with USP11 in the cytoplasm, and the recruitment of RRM1 to LaminB1 induced by ionizing radiation (IR) facilitates the binding of USP11 to the nuclear pore complex (NPC), promoting USP11 entry into the nucleus. Upon nuclear translocation, USP11 binds to E2F1 and inhibits the ubiquitin-mediated degradation of E2F1, thereby enhancing the transcriptional expression of RAD51AP1. Moreover, a specific RRM1 mutant lacking amino acids 731–793, crucial for its interaction with USP11 and recruitment to LaminB1, exhibits a dominant-negative effect on RAD51AP1 expression and HR. Truncations of RRM1 fail to inhibit the ubiquitin-mediated degradation of E2F1 and cannot promote the E2F1-mediated transactivation of RAD51AP1. Lastly, the full length of RRM1, not truncations, enhances tumor cells’ sensitivity to IR, underscoring its importance in radiotherapy resistance. Collectively, our results suggest a novel function of RRM1 in promoting HR-mediated DSB repair through positive regulation of RAD51AP1 transcription by direct interaction with USP11 and promoting subsequent USP11-mediated deubiquitination of E2F1. Our findings elucidate a previously unknown mechanism whereby RRM1 promotes HR-mediated DNA repair, presenting a potential therapeutic target for cancer treatment.

CBFβ Regulates RUNX3 ADP-Ribosylation to Mediate Homologous Recombination Repair.

RUNX3 is a master developmental transcriptional factor that has been implicated as a tumor suppressor in many cancers. However, the exact role of RUNX3 in cancer pathogenesis remains to be completely elucidated. Recently, it has emerged that RUNX3 is involved in the DNA damage response. Here, we demonstrate that heterodimerization of RUNX3 with CBFβ is necessary for its stability by protecting RUNX3 from RUNX3 ADP-ribosylation-dependent ubiquitination and degradation. We further identify new amino acid residues that are targets for PARylation and demonstrate that RUNX3 PARylation at these residues is necessary for localization of RUNX3 to DNA double strand break sites (DBSs). We also demonstrate that both RUNX3 PARylation and CBFβ heterodimerization with RUNX3 positively regulates homologous recombination (HR) repair, in part by promoting the recruitment of CtIP and phospho-RPA2 to the DBSs to mediate HR repair. In summary, we provide evidence that RUNX3 regulates HR repair activity in a PARylation-dependent manner.

GATA1 activates HSD17B6 to improve efficiency of cisplatin in lung adenocarcinoma via DNA damage

BackgroundLung adenocarcinoma (LUAD) is the most common histological type of non-small cell lung cancer (NSCLC). Platinum-based chemotherapy, such as cisplatin chemotherapy, is the cornerstone of treatment for LUAD patients. Nevertheless, cisplatin resistance remains the key obstacle to LUAD treatment, for its mechanism has not been fully elucidated.MethodsHSD17B6 mRNA expression data were accessed from TCGA-LUAD database and differential expression analysis was performed. Enrichment analysis of HSD17B6 was conducted by GSEA, and its upstream transcription factors were predicted by hTFtarget. mRNA and protein expression levels of HSD17B6 and GATA1 were assayed by qRT-PCR and WB, and the binding relationship between them was verified by chromatin immunoprecipitation assay and dual luciferase reporter assay. Cell viability and IC50 value of cisplatin-treated cells were measured by cell counting kit-8 assay. Cell cycle was assayed by flow cytometry. DNA damage level and DNA damage marker γ-H2AX expression were assayed by comet assay and western blot, respectively.ResultsHSD17B6 was lowly expressed in LUAD tissues and cells and mainly enriched in homologous recombination and mismatch repair pathways. As cell function experiments revealed, overexpression of HSD17B suppressed malignant phenotypes and cisplatin resistance in LUAD cells through DNA damage. Bioinformatics analysis revealed that GATA1 is the upstream regulator of HSD17B6, which was markedly reduced in LUAD tissues and cells. ChIP and dual luciferase reporter assays ascertained the binding of GATA1 to HSD17B6. Knockdown of GATA1 attenuated the effect of overexpression of HSD17B6 on LUAD cell behaviors and cisplatin resistance.ConclusionTranscription factor GATA1 could activate HSD17B6 to inhibit cisplatin resistance in LUAD through DNA damage, suggesting that GATA1/HSD17B6 axis may be a potential therapeutic target for chemotherapy resistance in LUAD patients.

Patterns of genomic instability in > 2000 patients with ovarian cancer across six clinical trials evaluating olaparib

BackgroundThe introduction of poly(ADP-ribose) polymerase (PARP) inhibitors represented a paradigm shift in the treatment of ovarian cancer. Genomic data from patients with high-grade ovarian cancer in six phase II/III trials involving the PARP inhibitor olaparib were analyzed to better understand patterns and potential causes of genomic instability.Patients and methodsHomologous recombination deficiency (HRD) was assessed in 2147 tumor samples from SOLO1, PAOLA-1, Study 19, SOLO2, OPINION, and LIGHT using next-generation sequencing technology. Genomic instability scores (GIS) were assessed in BRCA1 and/or BRCA2 (BRCA)-mutated (BRCAm), non-BRCA homologous recombination repair-mutated (non-BRCA HRRm), and non-HRRm tumors.ResultsBRCAm was identified in 1021/2147 (47.6%) tumors. BRCAm tumors had significantly higher GIS than non-BRCAm tumors (P < 0.001) and high biallelic loss (815/838; 97.3%) regardless of germline (658/672; 97.9%) or somatic (101/108; 93.5%) BRCAm status. In non-BRCA HRRm tumors (n = 121) a similar proportion were HRD-positive (GIS ≥ 42: 55/121; 45.5%) relative to HRD-negative (GIS < 42: 52/121; 43.0%). GIS was highly variable in non-BRCA HRRm (median 42 [interquartile range (IQR) 29–58]) and non-HRRm (n = 1005; median 32 [IQR 20–55]) tumors. Gene mutations with high GIS included HRR genes BRIP1 (median 46 [IQR 41–58]), RAD51C (median 58 [IQR 48–66]), RAD51D (median 62 [IQR 54–69]), and PALB2 (median 64 [IQR 58–74]), and non-HRR genes NF1 (median 49 [IQR 25–60]) and RB1 (median 55 [IQR 30–71]). CCNE1-amplified and PIK3CA-mutated tumors had low GIS (CCNE1-amplified: median 24 [IQR 18–29]; PIK3CA-mutated: median 32 [IQR 14–52]) and were predominantly non-BRCAm.ConclusionsThese analyses provide valuable insight into patterns of genomic instability and potential drivers of HRD, besides BRCAm, in ovarian cancer and will help guide future research into the potential clinical effectiveness of anti-cancer treatments in ovarian cancer, including PARP inhibitors as well as other precision oncology agents.Trial registrationThe SOLO1 trial was registered at ClinicalTrials.gov (NCT01844986) on April 30, 2013; the PAOLA-1 trial was registered at ClinicalTrials.gov (NCT02477644) on June 18, 2015 (retrospectively registered); Study 19 was registered at ClinicalTrials.gov (NCT00753545) on September 12, 2008 (retrospectively registered); the SOLO2 trial was registered at ClinicalTrials.gov (NCT01874353) on June 7, 2013; the OPINION trial was registered at ClinicalTrials.gov (NCT03402841) on January 3, 2018; the LIGHT trial was registered at ClinicalTrials.gov (NCT02983799) on November 4, 2016.

PRMT5-regulated splicing of DNA repair genes drives chemoresistance in breast cancer stem cells.

Breast cancer stem cells (BCSCs) are a rare cell population that is responsible for tumour initiation, metastasis and chemoresistance. Despite this, the mechanism by which BCSCs withstand genotoxic stress is largely unknown. Here, we uncover a pivotal role for the arginine methyltransferase PRMT5 in mediating BCSC chemoresistance by modulating DNA repair efficiency. Mechanistically, we identify PRMT5 as a major regulator of DNA damage response (DDR) gene splicing in BCSCs, particularly those integral to the Fanconi Anaemia and homologous recombination pathways, with PRMT5 inhibition synergising with chemotherapy to promote BCSC apoptosis. A comparison of BCSCs and their bulk cell progeny identified some shared (ATM, DDX11, EXO1, FAN1, SLX4) but many unique (ATR, RAD17, RAD51D, RUVBL1) PRMT5-dependent alternative DDR splicing events. Surprisingly, these skipped exons and retained intron events rarely lead to substantial gene expression repression, suggesting that PRMT5 inhibition predominantly results in nuclear detention of intron-containing transcripts and the production of non-canonical isoforms with compromised protein function. Since many genes within the same DDR pathway undergo deregulated splicing, this study thus reveals additional points of vulnerability and alternative combination drug strategies that could improve the therapeutic efficacy of PRMT5 inhibitors to promote BCSC eradication.

Molecular Subgroups of HRD Positive Ovarian Cancer and Their Prognostic Significance

Homologous recombination repair deficiency (HRD) is involved in the development of high-grade serous ovarian carcinoma (HGSOC) and its elevated sensitivity to platinum-based chemotherapy. To investigate the heterogeneity of the HRD-positive HGSOC we evaluated the HRD status, including BRCA mutations, genomic scar score, and methylation status of BRCA1/2 genes in 352 HGSOC specimens. We then divided the HRD-positive cohort into three molecular subgroups, the BRCA mutation cohort (BRCA+), BRCA1 methylation cohort (Meth+), and the rest of the HRD+ cohort (HRD+BRCA-Meth-), and evaluated their first-line chemotherapy response, benefit from olaparib, and progression-free survival (PFS). HRD-positive status was detected in 65% (228/352) of samples. The first group, BRCA+, accounted for 45% (102/228) of HRD positive cases and showed the best outcome in platinum therapy (ORR 96%), the highest olaparib benefit (p = 0.006) and the highest median PFS (46 months). The frequency of the second cohort, Meth+, among HRD-positive patients was 23% (52/228). Patients with Meth+ HGSOC showed a significantly poorer outcome, with a median PFS of 19 months, a significantly lower ORR to platinum therapy (84%) and a modest, but not significant, benefit from olaparib maintenance. The third HRD+BRCA-Meth- group accounted for 32% (74/228) of HRD-positive patients and showed an ORR to platinum therapy similar to that of the BRCA+ group (90%), a higher, but not statistically significant, benefit from olaparib and a median PFS of 23 months. In conclusion, Meth+ subgroup had poor outcomes in terms of chemotherapy response, olaparib benefit, and PFS compared to the other HRD+ subgroups, requiring a more thorough follow-up.

Minimizing DNA trapping while maintaining activity inhibition via selective PARP1 degrader

Poly (ADP-ribose) polymerase 1 (PARP1) catalyzes poly (ADP) ribosylation reaction, one of the essential post-translational modifications of proteins in eukaryotic cells. Given that PARP1 inhibition can lead to synthetic lethality in cells with compromised homologous recombination, this enzyme has been identified as a potent target for anti-cancer therapeutics. However, the clinical application of existing PARP1 inhibitors is restrained by side effects associated with DNA trapping and off-target effects, highlighting the need for improved therapeutic strategies. By integrating protein degradation technology, we synthesized a PROTAC molecule 180055 based on the Rucaparib junction and VHL ligand, which efficiently and selectively degraded PARP1 and inhibited PARP1 enzyme activity without a noticeable DNA trapping effect. Furthermore, 180055 kills tumor cells carrying BRCA mutations with a minor impact on the growth of normal cells both in vitro and in vivo. This suggests that 180055 is a PARP1-degrading compound with excellent pharmacological efficacy and extremely high biological safety that deserves further exploration and validation in clinical trials.

PARP1 Inhibitors: An Important Part in Cancer Treatment

Abstract: Cancer is a highly fatal disease that is typified by aberrant cells proliferating out of control. The properties of acquired cells, including as genomic instability and mutations, cellular death evasion, and proliferative signaling sustenance, are evidenced by the hallmarks of cancer and contribute to the establishment of malignant tumors. A protein called poly(ADP-ribose) polymerase- 1 (PARP1) is essential for both cell survival and DNA damage repair, two processes that affect cellular control. Since homologous recombination deficient cells exhibit cellular death upon suppression of PARP1, the PARP protein has gained attention as a potential target for anticancer treatments. The Food and Drug Administration (FDA) has already approved a number of effective PARP1 inhibitors, including olaparib and niraparib. The final chemical has a 1,3,4- thiadiazole core in its structure. In fact, heterocyclic moieties have gained attention due to their numerous medicinal advantages, which include their capacity to combat cancer. The compounds derived from these substances have been studied as PARP1 inhibitors, with promising results. Thus, the goal of this study is to go over the importance of PARP1 in cancer as well as its role in cell regulation. It also attempts to offer a comprehensive assessment of the literature that has been published in the previous fifteen years about PARP1 inhibitors, including the different scaffolds, with an emphasis on features of the structure-activity relationship. This will provide vital information for the creation of fresh, more effective PARP1 inhibitors.

Molecular Genetic and Clinical Characteristics of Fetuses With Chromosome 16 Short-Arm Microdeletions/Microduplications.

The short arm of chromosome 16 is highly susceptible to homologous recombination through nonallelic genes. This results in microdeletions/microduplications that can lead to neurodevelopmental disorders. However, incomplete penetrance and phenotypic diversity after birth exacerbate the uncertainty in prenatal genetic counseling. A total of 24,000 cases with prenatal diagnoses were retrospectively analyzed. Chromosome microarray analysis (CMA) was performed on 17,000 cases, of which 81 (0.48%) had chromosome 16 short-arm microdeletions/microduplications. Of the 81 fetuses with chromosome 16 short-arm microdeletions/microduplications, 36 and 28 had 16p11.2 and 16p13.11 microdeletions/microduplications, respectively. Ten, four, and three fetuses had 16p12.2, 16p13.12p13.11, and 16p13.12p1.3 microdeletions, respectively. Among the 36 fetuses with 16p11.2 microdeletions/microduplications, 33 had abnormal intrauterine ultrasound phenotypes, the most common being skeletal system abnormalities. Among the 28 fetuses with 16p13.11 microdeletions/microduplications, 19 had abnormal intrauterine ultrasound phenotypes, including 15 with abnormal ultrasonic soft markers. Among the 10 fetuses with the 16p12.2 microdeletions, six had abnormal ultrasound findings, and four had skeletal system abnormalities. After genetic counseling, 44 patients were selected and tested for family verification, of which 22 were de novo, while 22 were inherited from phenotypically normal parents. Among the 47 live births, 39 had no abnormalities. All fetuses with the 16p13.11 microdeletions/microduplications, and 16p12.2, 16p13.12p13.11, and 16p13.12p1.3 microdeletions were healthy after birth. Hence, chromosome 16 short-arm microdeletions/microduplications should not be the sole basis for abandoning pregnancy, and clinicians should consider prenatal diagnostic data to maximize diagnostic accuracy.

Role of BRCA1 in glioblastoma etiology.

BRCA1 (Breast Cancer 1) is a tumor suppressor gene with a role in DNA repair by Homologous Recombination (HR), and maintenance of genomic stability that is frequently investigated in breast, prostate, and ovarian cancers. BRCA1 mutations or dysregulation in glioblastoma can lead to impaired DNA repair mechanisms, resulting in tumor progression and resistance to standard therapies. Several studies have shown that BRCA1 expression is altered, albeit rarely, in glioblastoma, leading to poor prognosis and increased tumor aggressiveness. In addition, the communication of BRCA1 with other molecular pathways such as p53 and PTEN further complicates its role in glioblastoma pathogenesis. Targeting BRCA1-related pathways in these cases has shown the potential to improve the efficacy of standard treatments, including radiotherapy and chemotherapy. The development of (Poly (ADP-ribose) Polymerase) PARP inhibitors that exploit the lack of HR also offers a therapeutic approach to glioblastoma patients with BRCA1 mutations. Despite these advances, the heterogeneity of glioblastoma and its complex tumor microenvironment make the translation of such approaches into clinical practice still challenging, and there is an "unmet need". This review discusses the current mechanisms of etiology and potential treatment of BRCA1-related glioblastoma.

Research progress on pathogenic germline mutations in malignant tumors

Malignant tumors are closely related to various genetic and environmental factors. Pathogenic germline gene mutations play a key role in the occurrence and development of some malignant tumors. Some germline mutations can increase the risk of malignant tumors. For example, those with homologous recombination repair gene BRCA1/2 mutations are prone to breast cancer, ovarian cancer, etc., and some germline mutations are associated with genetic syndromes. For instance, 80% of hereditary non-polyposis colon cancers are associated with mutations in mismatch repair genes such as MLH1 and MLH2. In addition, 70% of Li-Fraumeni syndrome patients have harbored germline TP53 mutations. With the development of next-generation sequencing technology, more and more germline gene mutations have been discovered recently, which is of great significance for the prevention, screening, and treatment of tumors. This article has provided a review for common germline mutations, detection methods, and advances in drug therapy.

Talazoparib plus enzalutamide versus olaparib plus abiraterone acetate and niraparib plus abiraterone acetate for metastatic castration-resistant prostate cancer: a matching-adjusted indirect comparison.

Without head-to-head trials between talazoparib+enzalutamide (TALA + ENZA), olaparib+abiraterone acetate (OLAP + AAP), and niraparib plus AAP (NIRA + AAP) the ability to evaluate their relative efficacy as first-line (1 L) treatment in metastatic castration-resistant prostate cancer (mCRPC) is limited. The objective of this study was to assess the relative efficacy between TALA + ENZA (TALAPRO-2) versus OLAP + AAP (PROpel) and NIRA + AAP (MAGNITUDE) in 1 L mCRPC via a matching-adjusted indirect treatment comparison (MAIC). Patient-level data from TALAPRO-2 and published data from PROpel and MAGNITUDE were used. TALAPRO-2 data were reweighted to satisfy the eligibility criteria for PROpel and MAGNITUDE. Talazoparib (0.5 mg/day) plus enzalutamide (160 mg/day) was compared to olaparib (300 mg twice daily) plus abiraterone acetate (1000 mg/day) and niraparib (200 mg/day) plus abiraterone acetate (1000 mg/day). Hazard ratios (HRs) were calculated for radiographic progression-free survival (rPFS) and overall survival (OS), and odds ratios (ORs) for prostate-specific antigen (PSA) response and objective response rate (ORR). Additional efficacy outcomes were assessed. In all-comers, TALA + ENZA was statistically superior to OLAP + AAP for rPFS (HR: 0.727; 95% confidence interval [CI]: 0.565, 0.935) and PSA response (OR: 1.663; 1.101, 2.510), and numerically favored for OS (HR: 0.847; 0.667, 1.076) and ORR (OR: 1.109; 0.646, 1.903). In patients with homologous recombination repair mutations (HRRm), relative to NIRA + AAP, TALA + ENZA was statistically superior for rPFS (HR: 0.460; 0.280, 0.754), and numerically favored for OS (HR: 0.601; 0.347, 1.041) and ORR (OR: 1.524; 0.579, 4.016). Results suggest that TALA + ENZA may provide improvements in clinical outcomes relative to OLAP + AAP and NIRA + AAP in 1 L mCRPC; however, inherent limitations associated with the complexity of the analyses must be considered.

Beginning at the ends: telomere and telomere-based cancer therapeutics.

Telomeres, which are situated at the terminal ends of chromosomes, undergo a reduction in length with each cellular division, ultimately reaching a critical threshold that triggers cellular senescence. Cancer cells circumvent this senescence by utilizing telomere maintenance mechanisms (TMMs) that grant them a form of immortality. These mechanisms can be categorized into two primary processes: the reactivation of telomerase reverse transcriptase and the alternative lengthening of telomeres (ALT) pathway, which is dependent on homologous recombination (HR). Various strategies have been developed to inhibit telomerase activation in 85-95% of cancers, including the use of antisense oligonucleotides such as small interfering RNAs and endogenous microRNAs, agents that simulate telomere uncapping, expression modulators, immunotherapeutic vaccines targeting telomerase, reverse transcriptase inhibitors, stabilization of G-quadruplex structures, and gene therapy approaches. Conversely, in the remaining 5-15% of human cancers that rely on ALT, mechanisms involve modifications in the chromatin environment surrounding telomeres, upregulation of TERRA long non-coding RNA, enhanced activation of the ataxia telangiectasia and Rad-3-related protein kinase signaling pathway, increased interactions with nuclear receptors, telomere repositioning driven by HR, and recombination events between non-sister chromatids, all of which present potential targets for therapeutic intervention. Additionally, combinatorial therapy has emerged as a strategy that employs selective agents to simultaneously target both telomerase and ALT, aiming for optimal clinical outcomes. Given the critical role of anti-TMM strategies in cancer treatment, this review provides an overview of the latest insights into the structure and function of telomeres, their involvement in tumorigenesis, and the advancements in TMM-based cancer therapies.

Saccharomyces cerevisiae Rev7 promotes non-homologous end-joining by blocking Mre11 nuclease and Rad50’s ATPase activities and homologous recombination

Recent studies have shown that, in human cancer cells, the tetrameric Shieldin complex (comprising REV7, SHLD1, SHLD2, and SHLD3) facilitates non-homologous end-joining (NHEJ) while blocking homologous recombination (HR). Surprisingly, several eukaryotic species lack SHLD1, SHLD2, and SHLD3 orthologs, suggesting that Rev7 may leverage an alternative mechanism to regulate the double-strand break (DSB) repair pathway choice. Exploring this hypothesis, we discovered that Saccharomyces cerevisiae Rev7 physically interacts with the Mre11–Rad50–Xrs2 (MRX) subunits, impedes G-quadruplex DNA synergized HU-induced toxicity, and facilitates NHEJ, while antagonizing HR. Notably, we reveal that a 42-amino acid C-terminal fragment of Rev7 binds to the subunits of MRX complex, protects rev7∆ cells from G-quadruplex DNA-HU-induced toxicity, and promotes NHEJ by blocking HR. By comparison, the N-terminal HORMA domain, a conserved protein–protein interaction module, was dispensable. We further show that the full-length Rev7 impedes Mre11 nuclease and Rad50’s ATPase activities without affecting the latter’s ATP-binding ability. Combined, these results provide unanticipated insights into the functional interaction between the MRX subunits and Rev7 and highlight a previously unrecognized mechanism by which Rev7 facilitates DSB repair via NHEJ, and attenuation of HR, by blocking Mre11 nuclease and Rad50’s ATPase activities in S. cerevisiae.

Saccharomyces cerevisiae Rev7 promotes non-homologous end-joining by blocking Mre11 nuclease and Rad50's ATPase activities and homologous recombination.

Recent studies have shown that, in human cancer cells, the tetrameric Shieldin complex (comprising REV7, SHLD1, SHLD2, and SHLD3) facilitates non-homologous end-joining (NHEJ) while blocking homologous recombination (HR). Surprisingly, several eukaryotic species lack SHLD1, SHLD2, and SHLD3 orthologs, suggesting that Rev7 may leverage an alternative mechanism to regulate the double-strand break (DSB) repair pathway choice. Exploring this hypothesis, we discovered that Saccharomyces cerevisiae Rev7 physically interacts with the Mre11-Rad50-Xrs2 (MRX) subunits, impedes G-quadruplex DNA synergized HU-induced toxicity, and facilitates NHEJ, while antagonizing HR. Notably, we reveal that a 42-amino acid C-terminal fragment of Rev7 binds to the subunits of MRX complex, protects rev7∆ cells from G-quadruplex DNA-HU-induced toxicity, and promotes NHEJ by blocking HR. By comparison, the N-terminal HORMA domain, a conserved protein-protein interaction module, was dispensable. We further show that the full-length Rev7 impedes Mre11 nuclease and Rad50's ATPase activities without affecting the latter's ATP-binding ability. Combined, these results provide unanticipated insights into the functional interaction between the MRX subunits and Rev7 and highlight a previously unrecognized mechanism by which Rev7 facilitates DSB repair via NHEJ, and attenuation of HR, by blocking Mre11 nuclease and Rad50's ATPase activities in S. cerevisiae.

New Therapeutic Options for BRCA Mutant Patients.

Pathogenic variants in BRCA1 and BRCA2 are associated with significantly elevated lifetime risks of breast, ovarian, pancreatic, and prostate cancer. These genes are critical in double-strand break repair through homologous recombination. An understanding of the biology of BRCA1 and BRCA2 led to the development of targeted therapeutics, specifically poly(ADP-ribose) polymerase (PARP) inhibitors, which are approved by the US Food and Drug Administration for multiple BRCA1/2-associated cancers. Here, we discuss the development of PARP inhibitors, mechanisms of resistance, and the potential utility of these drugs beyond canonical BRCA1/2 tumors, and we describe novel agents under study.

Construction and Isolation of Recombinant Vaccinia Virus by Homologous Recombination Using Fluorescent Protein Markers.

Genetic modification of vaccinia virus (VACV) is a fundamental and valuable research technique in elucidating the function of VACV genes, as well as the development as vaccine vectors for other infectious diseases, oncolytic therapeutics for cancers, and protein expression systems in mammalian cells. Because of the large size of poxvirus genome and noninfectious feature of the naked viral DNA, construction of recombinant VACV relies on intracellular homologous recombination between transfected DNA and replicating viral DNA in infected cells occurred in VACV infected cells. The efficiency of homologous recombination event for vaccinia virus is relatively low, and recombinant viruses only account for 0.1% of progeny viruses. Therefore, fluorescent protein markers are often included in the transfected DNA to facilitate the selection and screening of recombined viruses. Here we provide a detailed procedure for the design, generation, isolation, and detection of recombinant VACV by homologous recombination using fluorescent protein markers.

Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.

A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N 6 methylated adenosines (m6A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.

Spatial immune heterogeneity in a mouse tumor model after immunotherapy.

Cancer immunotherapy is increasingly used in clinical practice, but its success rate is reduced by tumor escape from the immune system. This may be due to the genetic instability of tumor cells, which allows them to adapt to the immune response and leads to intratumoral immune heterogeneity. The study investigated spatial immune heterogeneity in the tumor microenvironment and its possible drivers in a mouse model of tumors induced by human papillomaviruses (HPV) following immunotherapy. Gene expression was determined by RNA sequencing and mutations by whole exome sequencing. A comparison of different tumor areas revealed heterogeneity in immune cell infiltration, gene expression, and mutation composition. While the mean numbers of mutations with every impact on gene expression or protein function were comparable in treated and control tumors, mutations with high or moderate impact were increased after immunotherapy. The genes mutated in treated tumors were significantly enriched in genes associated with ECM metabolism, degradation, and interactions, HPV infection and carcinogenesis, and immune processes such as antigen processing and presentation, Toll-like receptor signaling, and cytokine production. Gene expression analysis of DNA damage and repair factors revealed that immunotherapy upregulated Apobec1 and Apobec3 genes and downregulated genes related to homologous recombination and translesion synthesis. In conclusion, this study describes the intratumoral immune heterogeneity, that could lead to tumor immune escape, and suggests the potential mechanisms involved.