Repair Of Double-strand DNA Breaks Research Articles

Shaping the BRCAness mutational landscape by alternative double-strand break repair, replication stress and mitotic aberrancies.

Tumours with mutations in the BRCA1/BRCA2 genes have impaired double-stranded DNA break repair, compromised replication fork protection and increased sensitivity to replication blocking agents, a phenotype collectively known as ‘BRCAness’. Tumours with a BRCAness phenotype become dependent on alternative repair pathways that are error-prone and introduce specific patterns of somatic mutations across the genome. The increasing availability of next-generation sequencing data of tumour samples has enabled identification of distinct mutational signatures associated with BRCAness. These signatures reveal that alternative repair pathways, including Polymerase θ-mediated alternative end-joining and RAD52-mediated single strand annealing are active in BRCA1/2-deficient tumours, pointing towards potential therapeutic targets in these tumours. Additionally, insight into the mutations and consequences of unrepaired DNA lesions may also aid in the identification of BRCA-like tumours lacking BRCA1/BRCA2 gene inactivation. This is clinically relevant, as these tumours respond favourably to treatment with DNA-damaging agents, including PARP inhibitors or cisplatin, which have been successfully used to treat patients with BRCA1/2-defective tumours. In this review, we aim to provide insight in the origins of the mutational landscape associated with BRCAness by exploring the molecular biology of alternative DNA repair pathways, which may represent actionable therapeutic targets in in these cells.

Meiotic DNA break repair can utilize homolog-independent chromatid templates in C. elegans

During meiosis, the maintenance of genome integrity is critical for generating viable haploid gametes.1 In meiotic prophase I, double-strand DNA breaks (DSBs) are induced and a subset of these DSBs are repaired as interhomolog crossovers to ensure proper chromosome segregation. DSBs not resolved as crossovers with the homolog must be repaired by other pathways to ensure genome integrity.2 To determine if alternative repair templates can be engaged for meiotic DSB repair during oogenesis, we developed an assay to detect sister and/or intra-chromatid repair events at a defined DSB site during Caenorhabditis elegans meiosis. Using this assay, we directly demonstrate that the sister chromatid or the same DNA molecule can be engaged as a meiotic repair template for both crossover and noncrossover recombination, with noncrossover events being the predominant recombination outcome. We additionally find that the sister or intra-chromatid substrate is available as a recombination partner for DSBs induced throughout meiotic prophase I, including late prophase when the homolog is unavailable. Analysis of noncrossover conversion tract sequences reveals that DSBs are processed similarly throughout prophase I. We further present data indicating that the XPF-1 nuclease functions in late prophase to promote sister or intra-chromatid repair at steps of recombination following joint molecule processing. Despite its function in sister or intra-chromatid repair, we find that xpf-1 mutants do not exhibit severe defects in progeny viability following exposure to ionizing radiation. Overall, we propose that C.elegans XPF-1 may assist as an intersister or intrachromatid resolvase only in late prophase I.

Direct unfolding of RuvA-HJ complex at the single-molecule level

The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (Funfold) of the HJ by ∼2-fold. Compared with the ΔGunfold of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.

Pattern of RAD51-G135C Polymorphism among Eastern Mediterranean Turkish Population and Association with Breast Cancer

The RAD51 gene encodes proteins that are important for the repair of double-strand DNA breaks by recombination. Thus, genetic variability in the gene may contribute to the occurrence and progression of breast carcinoma. We investigated the association of polymorphisms in the DNA repair genes RAD51-site 135G/C with the breast cancer risk. Genotypes were determined by PCR-RFLP assays in 55 female patients with breast cancers and 80 age-matched healthy controls. Bloods samples were taken from all the patients and health controls and DNA was isolated. The region of interest was amplified based on PCR. After amplification, we used a restriction enzyme (RAD51; MvaI) and digested the PCR product. Then, these DNA fragments were size separated in gel electrophoresis. We identified changes in the nucleotides in these specific regions and confirmed the genotyping via sequencing a subset of PCR products using an Applied Biosystems Automated Sequencer. We observed that both allele and genotype frequencies significantly differed between breast cancer patients and healthy control. The frequency of variant allele, C, was increased to 0.25 in breast cancer group as opposed to the frequency of 0.125 in control group. Similarly, the genotypes carrying the variant allele C (CC + GC) had an elevated frequency and increased odd’s ratio among breast cancer group. The obtained results indicate that the polymorphism of RAD51 genes may be associated with the incidence of breast cancer in the Eastern Mediterranean region population. We hypothesized that common polymorphisms in DNA repair and cell cycle regulator genes modify DNA repair machinery which contribute to breast cancer susceptibility.

A Perspective of Epigenetic Regulation in Radiotherapy.

Radiation therapy (RT) has been employed as a tumoricidal modality for more than 100 years and on 470,000 patients each year in the United States. The ionizing radiation causes genetic changes and results in cell death. However, since the biological mechanism of radiation remains unclear, there is a pressing need to understand this mechanism to improve the killing effect on tumors and reduce the side effects on normal cells. DNA break and epigenetic remodeling can be induced by radiotherapy. Hence the modulation of histone modification enzymes may tune the radiosensitivity of cancer cells. For instance, histone deacetylase (HDAC) inhibitors sensitize irradiated cancer cells by amplifying the DNA damage signaling and inhibiting double-strand DNA break repair to influence the irradiated cells’ survival. However, the combination of epigenetic drugs and radiotherapy has only been evaluated in several ongoing clinical trials for limited cancer types, partly due to a lack of knowledge on the potential mechanisms on how radiation induces epigenetic regulation and chromatin remodeling. Here, we review recent advances of radiotherapy and radiotherapy-induced epigenetic remodeling and introduce related technologies for epigenetic monitoring. Particularly, we exploit the application of fluorescence resonance energy transfer (FRET) biosensors to visualize dynamic epigenetic regulations in single living cells and tissue upon radiotherapy and drug treatment. We aim to bridge FRET biosensor, epigenetics, and radiotherapy, providing a perspective of using FRET to assess epigenetics and provide guidance for radiotherapy to improve cancer treatment. In the end, we discuss the feasibility of a combination of epigenetic drugs and radiotherapy as new approaches for cancer therapeutics.

Kinase CDK2 in Mammalian Meiotic Prophase I: Screening for Hetero- and Homomorphic Sex Chromosomes.

Cyclin-dependent kinases (CDKs) are crucial regulators of the eukaryotic cell cycle. The critical role of CDK2 in the progression of meiosis was demonstrated in a single mammalian species, the mouse. We used immunocytochemistry to study the localization of CDK2 during meiosis in seven rodent species that possess hetero- and homomorphic male sex chromosomes. To compare the distribution of CDK2 in XY and XX male sex chromosomes, we performed multi-round immunostaining of a number of marker proteins in meiotic chromosomes of the rat and subterranean mole voles. Antibodies to the following proteins were used: RAD51, a member of the double-stranded DNA break repair machinery; MLH1, a component of the DNA mismatch repair system; and SUN1, which is involved in the connection between the meiotic telomeres and nuclear envelope, alongside the synaptic protein SYCP3 and kinetochore marker CREST. Using an enhanced protocol, we were able to assess the distribution of as many as four separate proteins in the same meiotic cell. We showed that during prophase I, CDK2 localizes to telomeric and interstitial regions of autosomes in all species investigated (rat, vole, hamster, subterranean mole voles, and mole rats). In sex bivalents following synaptic specificity, the CDK2 signals were distributed in three different modes. In the XY bivalent in the rat and mole rat, we detected numerous CDK2 signals in asynaptic regions and a single CDK2 focus on synaptic segments, similar to the mouse sex chromosomes. In the mole voles, which have unique XX sex chromosomes in males, CDK2 signals were nevertheless distributed similarly to the rat XY sex chromosomes. In the vole, sex chromosomes did not synapse, but demonstrated CDK2 signals of varying intensity, similar to the rat X and Y chromosomes. In female mole voles, the XX bivalent had CDK2 pattern similar to autosomes of all species. In the hamster, CDK2 signals were revealed in telomeric regions in the short synaptic segment of the sex bivalent. We found that CDK2 signals colocalize with SUN1 and MLH1 signals in meiotic chromosomes in rats and mole voles, similar to the mouse. The difference in CDK2 manifestation at the prophase I sex chromosomes can be considered an example of the rapid chromosome evolution in mammals.

Identification of a novel pathogenic variant in PALB2 and BARD1 genes by a multigene sequencing panel in triple negative breast cancer in Morocco.

Pathogenic variants (PVs) in BRCA genes have been mainly associated with an increasing risk of triple negative breast cancer (TNBC). The contribution of PVs in non-BRCA genes to TNBC seems likely since the processing of homologous recombination repair of double-strand DNA breaks involves several genes. Here, we investigate the susceptibility of genetic variation of the BRCA and non-BRCA genes in 30 early-onset Moroccan women with TNBC.Methods: Targeted capture-based next generation sequencing (NGS) method was performed with a multigene panel testing (MGPT) for variant screening. Panel sequencing was performed with genes involved in hereditary predisposition to cancer and candidate genes whose involvement remains unclear using Illumina MiSeq platform. Interpretation was conducted by following the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) criteria.Results: PVs were identified in 20% (6/30) of patients with TNBC. Of these, 16.7% (5/30) carried a BRCA PV [10% (3/30) in BRCA1, 6.7% (2/30) in BRCA2] and 6.6% (2/30) carried a non-BRCA PV. The identified PVs in BRCA genes (BRCA1 c.798_799delTT, BRCA1 c.3279delC, BRCA2 c.1310_1313del, and BRCA2 c.1658T>G) have been reported before and were classified as pathogenic. The identified founder PVs BRCA1 c.798_799del and BRCA2 c.1310_1313delAAGA represented 10% (3/30). Our MGPT allowed identification of several sequence variations in most investigated genes, among which we found novel truncating variations in PALB2 and BARD1 genes. The PALB2 c.3290dup and BARD1 c.1333G>T variants are classified as pathogenic. We also identified 42 variants of unknown/uncertain significance (VUS) in 70% (21/30) of patients with TNBC, including 50% (21/42) missense variants. The highest VUS rate was observed in ATM (13%, 4/30). Additionally, 35.7% (15/42) variants initially well-known as benign, likely benign or conflicting interpretations of pathogenicity have been reclassified as VUS according to ACMG-AMP.Conclusions: PALB2 and BARD1 along with BRCA genetic screening could be helpful for a larger proportion of early-onset TNBC in Morocco.

Involvement of DNA Repair Genes and System of Radiation-Induced Activation of Transposons in Formation of Transgenerational Effects.

The study of the genetic basis of the manifestation of radiation-induced effects and their transgenerational inheritance makes it possible to identify the mechanisms of adaptation and possible effective strategies for the survival of organisms in response to chronic radioactive stress. One persistent hypothesis is that the activation of certain genes involved in cellular defense is a specific response of the cell to irradiation. There is also data indicating the important role of transposable elements in the formation of radiosensitivity/radioresistance of biological systems. In this work, we studied the interaction of the systems of hobo transposon activity and DNA repair in the cell under conditions of chronic low-dose irradiation and its participation in the inheritance of radiation-induced transgenerational instability in Drosophila. Our results showed a significant increase of sterility and locus-specific mutability, a decrease of survival, fertility and genome stability (an increase the frequency of dominant lethal mutations and DNA damage) in non-irradiated F1/F2 offspring of irradiated parents with dysfunction of the mus304 gene which is responsible for excision and post-replicative recombination repair and repair of double-stranded DNA breaks. The combined action of dysfunction of the mus309 gene and transpositional activity of hobo elements also led to the transgenerational effects of irradiation but only in the F1 offspring. Dysfunction of the genes of other DNA repair systems (mus101 and mus210) showed no visible effects inherited from irradiated parents subjected to hobo transpositions. The mei-41 gene showed specificity in this type of interaction, which consists in its higher efficiency in sensing events induced by transpositional activity rather than irradiation.

CXCR4 and RIF1 overexpression induces resistance of epithelial ovarian cancer to cisplatin-based chemotherapy.

The chemoresistance of epithelial ovarian cancer (EOC) is a major problem. Thus, the search for novel biomarkers associated with cisplatin sensitivity is overwhelming. Previous studies have shown that CXC chemokine receptor 4 (CXCR4) is associated with tumor growth, angiogenesis, and distant metastases, and replication timing regulatory factor 1 (RIF1) is responsible for the repair of double-strand DNA breaks. This study, thus, aimed to identify the correlation between CXCR4 and RIF1 overexpression and cisplatin sensitivity in EOC. Fifty-five EOC patients were recruited to assess the chemosensitivity of EOC to cisplatin-based chemotherapy at the Oncology Department in Tanta University Hospitals, Egypt. The results showed that patients with a higher CXCR4 and RIF1 expression exhibited a significantly lower chemosensitivity, worse overall survival, and poorer progression-free survival. The only prognostic associated with overall survival was CXCR4. Our study showed that CXCR4 and RIF1expression levels are not associated only with poor prognostic features of epithelial ovarian cancer but also with its chemoresistance to cisplatin, and consequently, with worse overall survival and progression free survival.

DNA Repair Syndromes and Cancer: Insights Into Genetics and Phenotype Patterns.

DNA damage response is essential to human physiology. A broad spectrum of pathologies are displayed by individuals carrying monoallelic or biallelic loss-of-function mutations in DNA damage repair genes. DNA repair syndromes with biallelic disturbance of essential DNA damage response pathways manifest early in life with multi-systemic involvement and a high propensity for hematologic and solid cancers, as well as bone marrow failure. In this review, we describe classic biallelic DNA repair cancer syndromes arising from faulty single- and double-strand DNA break repair, as well as dysfunctional DNA helicases. These clinical entities include xeroderma pigmentosum, constitutional mismatch repair deficiency, ataxia telangiectasia, Nijmegen breakage syndrome, deficiencies of DNA ligase IV, NHEJ/Cernunnos, and ERCC6L2, as well as Bloom, Werner, and Rothmund-Thompson syndromes. To give an in-depth understanding of these disorders, we provide historical overview and discuss the interplay between complex biology and heterogeneous clinical manifestations.

Bridging of DNA breaks activates PARP2-HPF1 to modify chromatin.

Summary:DNA strand breaks recruit PARP1 and its paralogue PARP2 to modify histones and many other substrates with mono- and poly(ADP-ribose) (PAR)1–5. In DNA damage response, the PAR post-translational modification occurs predominantly on serine amino acids6–8, which requires HPF1, an accessory factor that switches the amino-acid specificity of PARP1/2 from aspartate/glutamate to serine residues9,10. Poly(ADP) ribosylation (PARylation) is important for subsequent chromatin decompaction and serves as an anchor to recruit a variety of downstream signaling and repair factors to the sites of DNA breaks2,11. To understand the molecular mechanism of DNA break recognition by PARP enzymes in the context of chromatin, we determined cryo-EM structure of PARP2/HPF1 bound to a nucleosome. The structure shows that PARP2/HPF1 bridges two nucleosomes, with the broken DNA aligned in a ligation-competent position, revealing the initial step in double-strand DNA break repair. The bridging induces structural changes in PARP2 that signal DNA break recognition to the catalytic domain, which licenses HPF1 binding and PARP2 activation. Our data suggest that active PARP2 cycles through different conformational states to exchange NAD+ and substrate, which may enable PARP enzymes to be processive while bound to chromatin. The mechanisms of PARP activation and catalytic cycle we describe can explain resistance mechanisms to PARP inhibitors, and will aid development of better inhibitors for cancer treatments12–16.

Investigation of Break-Induced Replication in Yeast.

Repair of double-strand DNA breaks (DSBs) is important for preserving genomic integrity and stability. Break-induced replication (BIR) is a mechanism aimed to repair one-ended double-strand DNA breaks, similar to those formed by replication fork collapse or by telomere erosion. Unlike S-phase replication, BIR is carried out by a migrating DNA bubble and is associated with conservative inheritance of newly synthesized DNA. This unusual DNA synthesis leads to high level of mutagenesis and chromosomal rearrangements during BIR. Here, we focus on several genetic and molecular methods to investigate BIR using our system in yeast Saccharomyces cerevisiae where BIR is initiated by a site-specific DNA break, and the repair involves two copies of chromosome III.

Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity.

Cutaneous viral infections occur in a background of near continual exposure to environmental genotoxins, like UV radiation in sunlight. Failure to repair damaged DNA is an established driver of tumorigenesis and substantial cellular resources are devoted to repairing DNA lesions. Beta-human papillomaviruses (β-HPVs) attenuate DNA repair signaling. However, their role in human disease is unclear. Some have proposed that β-HPV promotes tumorigenesis, while others suggest that β-HPV protects against skin cancer. Most of the molecular evidence that β-HPV impairs DNA repair has been gained via characterization of the E6 protein from β-HPV 8 (β-HPV 8E6). Moreover, β-HPV 8E6 hinders DNA repair by binding and destabilizing p300, a transcription factor for multiple DNA repair genes. By reducing p300 availability, β-HPV 8E6 attenuates a major double strand DNA break (DSB) repair pathway, homologous recombination. Here, β-HPV 8E6 impairs another DSB repair pathway, non-homologous end joining (NHEJ). Specifically, β-HPV 8E6 acts by attenuating DNA-dependent protein kinase (DNA-PK) activity, a critical NHEJ kinase. This includes DNA-PK activation and the downstream of steps in the pathway associated with DNA-PK activity. Notably, β-HPV 8E6 inhibits NHEJ through p300 dependent and independent means. Together, these data expand the known genome destabilizing capabilities of β-HPV 8E6.

EIF3a Regulation of NHEJ Repair Protein Synthesis and Cellular Response to Ionizing Radiation.

Translation initiation in protein synthesis regulated by eukaryotic initiation factors (eIFs) is a crucial step in controlling gene expression. eIF3a has been shown to regulate protein synthesis and cellular response to treatments by anticancer agents including cisplatin by regulating nucleotide excision repair. In this study, we tested the hypothesis that eIF3a regulates the synthesis of proteins important for the repair of double-strand DNA breaks induced by ionizing radiation (IR). We found that eIF3a upregulation sensitized cellular response to IR while its downregulation caused resistance to IR. eIF3a increases IR-induced DNA damages and decreases non-homologous end joining (NHEJ) activity by suppressing the synthesis of NHEJ repair proteins. Furthermore, analysis of existing patient database shows that eIF3a expression associates with better overall survival of breast, gastric, lung, and ovarian cancer patients. These findings together suggest that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating the synthesis of DNA repair proteins and, thus, eIIF3a likely contributes to the outcome of cancer patients treated with DNA-damaging strategies including IR.

Abstract 3554: Genomic landscape of acquired uniparental disomy in NCI PDMR patient derived xenograft models

Abstract Background: Acquired Uniparental Disomy (aUPD) is relatively common in cancer. Occurrence of aUPD is more frequent in some tumor histologies (e.g., serous ovarian, colorectal) and may be relevant for choice of therapy. The Patient-Derived Models Repository (PDMR; https://pdmr.cancer.gov) developed by The National Cancer Institute (NCI) includes patient-derived xenograft (PDX) models from multiple tumor histologies with different passages and lineages. The associated clinical annotation and genomic data make it possible to assess the prevalence of aUPD in the PDMR cohort and the stability of aUPD in different passages and lineages within a PDX model. Methods: High tumor purity in the PDX specimens (after removal of mouse reads representing the stroma) enabled highly accurate assessment of loss of heterozygosity (LOH). Variants called by GATK Haplotype caller from whole exome sequencing (WES) data were used to identify segments of homozygosity using BCFtools/RoH (runs of homozygosity). The RoH segments were then intersected with the bed file for chromosome arms to get %LOH at the arm level. If %LOH on a chromosome arm was >90%, we considered the sample to have aUPD at the arm level. WES was also used to look for associations between DNA damage repair (DDR) pathway alterations and aUPD. Results: We made the following observations: a) aUPD was observed most frequently in chr18q (75/427, 17.6%) and chr3p (69/427, 16%) of PDX models; b) aUPD was observed more frequently in certain tumor histologies, e.g., clear cell renal cell carcinoma (6/8), small cell lung cancer (3/4) and non-small cell lung cancer (25/38); c) extensive aUPD was observed in 4 PDMR models (>50% of evaluated chromosome arms in these models have aUPD); d) aUPD was not observed in some tumor histologies, i.e., synovial sarcoma, uterine endometrioid carcinoma; e) in the vast majority of PDMR models (>90%), aUPD is maintained faithfully across lineages and through multiple passaging; f) subclonal aUPD events were observed in some models across different lineages; g) significant enrichment of double strand DNA break repair (DSBR) pathway alterations was observed in PDMR models without aUPD (p=0.0007, Fisher's exact test) suggesting defects in DSBR are not associated with aUPD; and h) aUPD was rarely observed in MSI-high models (1/30) suggesting mutual exclusivity of mismatch repair (MMR) pathway defects and aUPD. Conclusion: We observed a relatively high frequency of UPD in the PDMR models (at least 1 arm of a chromosome). UPD was more frequently observed in specific chromosomal arms. The frequency of aUPD was higher in some tumor histologies and absent in others. aUPD was stably maintained across passages and lineages, although some heterogeneity was observed. Our data suggest aUPD is not associated with defects in DSBR and MMR pathways. Preclinical drug studies using NCI PDMR models may suggest appropriate therapeutic options for cancers with aUPD. Citation Format: Rajesh Patidar, Li Chen, Chris A. Karlovich, Biswajit Das, Yvonne A. Evrard, Tomas Vilimas, Justine N. McCutcheon, Amanda L. Peach, Nikitha V. Nair, Thomas D. Forbes, Brandie A. Fullmer, Anna J. Lee Fong, Luis E. Romero, Alyssa K. Chapman, Kelsey A. Conley, Robin D. Harrington, Shahanawaz S. Jiwani, Peng Wang, Michelle M. Gottholm Ahalt, Erin N. Cantu, Gloryvee Rivera, Lindsay M. Dutko, Kelly M. Benauer, Vishnuprabha R. Kannan, Suzanne D. Borgel, John P. Carter, Jesse M. Stottlemyer, Tiffanie L. Miner, Devynn R. Breen, Emily T. Delaney, Chelsea A. McGlynn, Candace N. Mallow, Marianne Radzyminski, Shannon N. Uzelac, Sergio Y. Alcoser, Tara L. Grinnage-Pulley, Michelle A. Eugeni, Dianne L. Newton, Melinda G. Hollingshead, Paul M. Williams, James H. Doroshow. Genomic landscape of acquired uniparental disomy in NCI PDMR patient derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3554.

Abstract 3536: Genetic validation of an ATM immunohistochemistry assay for genomic and clinical-pathologic characterization of primary prostate cancers with ATM loss

Abstract Background: ATM, a protein kinase involved in double-strand DNA break repair, is mutated in a significant fraction of prostate cancers, and targeted therapies such as ATR inhibitors appear promising for ATM-deficient tumors. However, few ATM-deficient primary prostate cancers have been clinically and genomically characterized. Design: We validated an automated and dichotomously-scored clinical-grade immunohistochemistry (IHC) assay to detect ATM protein loss using prostate cancer cell lines and 52 very high-grade prostate tumors (Gleason score 5+4=9 or 5+5=10) with known ATM genomic status. We examined the frequency of ATM protein loss among 29 tumors with known pathogenic germline ATM mutations or variants of unknown significance (VUS). Finally, we screened more than 800 additional primary prostate carcinomas using tissue microarrays (TMA) for ATM protein loss. Targeted somatic genomic sequencing using HRD Plus (Myriad Genetics) or UW-OncoPlex was conducted in a subset of immunostained tumors in each cohort. Results: ATM protein loss by IHC was found in 13% (7/52) of very high-grade (primary Gleason pattern 5) tumors with somatic genomic sequencing data. Of 7 cases with ATM protein loss, 4 had a deleterious ATM mutation and 3 had apparent single copy ATM genomic loss. Of the remaining 45 cases without ATM protein loss, none had ATM genomic alterations. In the cohort of tumors with pathogenic germline ATM mutations, 74% (17/23) had ATM protein loss, but only 45% (9/20) with evaluable sequencing had a second pathogenic alteration in ATM detected (all of these had protein loss). Of those with germline mutations and ATM protein loss, the majority (76% or 13/17) had an apparent clonal alteration with homogeneous protein loss in all tumor cells sampled. In contrast, among 6 cases with germline ATM VUS, only one had protein loss. On TMA analysis, ATM loss was present in 3% (25/824) of evaluable tumors overall, and was significantly more common in tumors from Grade Group 5 (12/130; 9.2%) compared to all other Grade Groups (13/694; 1.9%) (P<0.0001). Of tumors found by TMA screening with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 58% (7/12) with heterogeneous ATM protein loss had detectable pathogenic alterations in ATM. Of these, 82% (9/11) were inferred to have somatic ATM variants. Conclusions: Validated ATM IHC is a sensitive assay for detecting underlying ATM alterations and may be useful to evaluate VUS in ATM. However, not all tumors with ATM protein loss have detectable genomic alterations. ATM protein loss is likely an early clonal event in tumors with germline mutations and is significantly enriched in high-grade prostate cancers. Citation Format: Harsimar Kaur, Jessica Hicks, Colin Pritchard, Angelo De Marzo, William Isaacs, Jerry Lanchbury, Kirsten Timms, Emmanuel Antonarakis, Tamara L. Lotan. Genetic validation of an ATM immunohistochemistry assay for genomic and clinical-pathologic characterization of primary prostate cancers with ATM loss [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3536.

The Chromatin Response to Double-Strand DNA Breaks and Their Repair.

Cellular DNA is constantly being damaged by numerous internal and external mutagenic factors. Probably the most severe type of insults DNA could suffer are the double-strand DNA breaks (DSBs). They sever both DNA strands and compromise genomic stability, causing deleterious chromosomal aberrations that are implicated in numerous maladies, including cancer. Not surprisingly, cells have evolved several DSB repair pathways encompassing hundreds of different DNA repair proteins to cope with this challenge. In eukaryotic cells, DSB repair is fulfilled in the immensely complex environment of the chromatin. The chromatin is not just a passive background that accommodates the multitude of DNA repair proteins, but it is a highly dynamic and active participant in the repair process. Chromatin alterations, such as changing patterns of histone modifications shaped by numerous histone-modifying enzymes and chromatin remodeling, are pivotal for proficient DSB repair. Dynamic chromatin changes ensure accessibility to the damaged region, recruit DNA repair proteins, and regulate their association and activity, contributing to DSB repair pathway choice and coordination. Given the paramount importance of DSB repair in tumorigenesis and cancer progression, DSB repair has turned into an attractive target for the development of novel anticancer therapies, some of which have already entered the clinic.

HIV Vpr Modulates the Host DNA Damage Response at Two Independent Steps to Damage DNA and Repress Double-Strand DNA Break Repair.

The DNA damage response (DDR) is a signaling cascade that is vital to ensuring the fidelity of the host genome in the presence of genotoxic stress. Growing evidence has emphasized the importance of both activation and repression of the host DDR by diverse DNA and RNA viruses. Previous work has shown that HIV-1 is also capable of engaging the host DDR, primarily through the conserved accessory protein Vpr. However, the extent of this engagement has remained unclear. Here, we show that HIV-1 and HIV-2 Vpr directly induce DNA damage and stall DNA replication, leading to the activation of several markers of double- and single-strand DNA breaks. Despite causing damage and activating the DDR, we found that Vpr represses the repair of double-strand breaks (DSB) by inhibiting homologous recombination (HR) and nonhomologous end joining (NHEJ). Mutational analyses of Vpr revealed that DNA damage and DDR activation are independent from repression of HR and Vpr-mediated cell cycle arrest. Moreover, we show that repression of HR does not require cell cycle arrest but instead may precede this long-standing enigmatic Vpr phenotype. Together, our data uncover that Vpr globally modulates the host DDR at at least two independent steps, offering novel insight into the primary functions of lentiviral Vpr and the roles of the DNA damage response in lentiviral replication.IMPORTANCE The DNA damage response (DDR) is a signaling cascade that safeguards the genome from genotoxic agents, including human pathogens. However, the DDR has also been utilized by many pathogens, such as human immunodeficiency virus (HIV), to enhance infection. To properly treat HIV-positive individuals, we must understand how the virus usurps our own cellular processes. Here, we have found that an important yet poorly understood gene in HIV, Vpr, targets the DDR at two unique steps: it causes damage and activates DDR signaling, and it represses the ability of cells to repair this damage, which we hypothesize is central to the primary function of Vpr. In clarifying these important functions of Vpr, our work highlights the multiple ways human pathogens engage the DDR and further suggests that modulation of the DDR is a novel way to help in the fight against HIV.

Role of RadA and DNA Polymerases in Recombination-Associated DNA Synthesis in Hyperthermophilic Archaea.

Among the three domains of life, the process of homologous recombination (HR) plays a central role in the repair of double-strand DNA breaks and the restart of stalled replication forks. Curiously, main protein actors involved in the HR process appear to be essential for hyperthermophilic Archaea raising interesting questions about the role of HR in replication and repair strategies of those Archaea living in extreme conditions. One key actor of this process is the recombinase RadA, which allows the homologous strand search and provides a DNA substrate required for following DNA synthesis and restoring genetic information. DNA polymerase operation after the strand exchange step is unclear in Archaea. Working with Pyrococcus abyssi proteins, here we show that both DNA polymerases, family-B polymerase (PolB) and family-D polymerase (PolD), can take charge of processing the RadA-mediated recombination intermediates. Our results also indicate that PolD is far less efficient, as compared with PolB, to extend the invaded DNA at the displacement-loop (D-loop) substrate. These observations coincide with previous genetic analyses obtained on Thermococcus species showing that PolB is mainly involved in DNA repair without being essential probably because PolD could take over combined with additional partners.

Half a century of bacteriophage lambda recombinase: In vitro studies of lambda exonuclease and Red-beta annealase.

DNA recombination, replication, and repair are intrinsically interconnected processes. From viruses to humans, they are ubiquitous and essential to all life on Earth. Single‐strand annealing homologous DNA recombination is a major mechanism for the repair of double‐stranded DNA breaks. An exonuclease and an annealase work in tandem, forming a complex known as a two‐component recombinase. Redβ annealase and λ‐exonuclease from phage lambda form the archetypal two‐component recombinase complex. In this short review article, we highlight some of the in vitro studies that have led to our current understanding of the lambda recombinase system. We synthesize insights from more than half a century of research, summarizing the state of our current understanding. From this foundation, we identify the gaps in our knowledge and cast an eye forward to consider what the next 50 years of research may uncover.