Unique Double-strand Break Research Articles

Abstract A018: Lung cancer associated DNA Damageome reveals a genome destabilizing role of Aquaporin 3

Abstract Lung cancer is a multifactorial disease caused by both environmental exposures and genetic factors. Genome and transcriptome-wide association studies (GWAS and TWAS) successfully identified many genetic variants and candidate causal genes associated with lung cancer. However, the lack of functional studies of these genes remains a major bottleneck to clinical translation. Recently, we discovered that large networks of evolutionarily conserved DNA damageome proteins (DDPs) promote DNA damage and genome instability. Here, we discovered a lung cancer-associated DNA damageome network. We further discovered that one of the lung cancer DDPs, Aquaporin 3 (AQP3), can promote ROS dependent DNA damage upon overproduction. Furthermore, it potentiates DNA damage by a low dose of arsenic exposure, creating unique double strand break hotspots which help predict mutational signatures. We screened for high endogenous DNA damage levels for more than 40 TWAS and GWAS-nominated candidates using high-throughput single cell-based flow cytometric assays. We optimized and performed Mre11-Cut&Tag (Cleavage Under Targets and Tagmentation) to map double strand breaks, which are the most deleterious form of DNA damage. In addition, we used END-Seq to provide a much higher resolution of the DSB map and uncovered hotspots in arsenic-detoxifying genes. We are implementing duplex sequencing to map the mutational signatures caused by arsenic and the arsenic-Aquaporin 3 interaction. Our study brings function to endogenous DNA damage and DNA damageome proteins that interact with environmental toxicants. Mechanistic insights into how low-dose arsenic interacts with risk genes yield critical knowledge for the prevention, diagnosis, and treatment of arsenic-associated diseases. We also uncovered several early biomarkers to potentially predict the long-term health impacts of arsenic. Citation Format: Jun Xia, Mu Lin, Zhuoyi Song, Gail Fernandes, Susan Rosenberg, Chris Amos. Lung cancer associated DNA Damageome reveals a genome destabilizing role of Aquaporin 3 [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A018.

Single molecule microscopy reveals key physical features of repair foci in living cells.

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

Members of the RAD52 Epistasis Group Contribute to Mitochondrial Homologous Recombination and Double-Strand Break Repair in Saccharomyces cerevisiae.

Mitochondria contain an independently maintained genome that encodes several proteins required for cellular respiration. Deletions in the mitochondrial genome have been identified that cause several maternally inherited diseases and are associated with certain cancers and neurological disorders. The majority of these deletions in human cells are flanked by short, repetitive sequences, suggesting that these deletions may result from recombination events. Our current understanding of the maintenance and repair of mtDNA is quite limited compared to our understanding of similar events in the nucleus. Many nuclear DNA repair proteins are now known to also localize to mitochondria, but their function and the mechanism of their action remain largely unknown. This study investigated the contribution of the nuclear double-strand break repair (DSBR) proteins Rad51p, Rad52p and Rad59p in mtDNA repair. We have determined that both Rad51p and Rad59p are localized to the matrix of the mitochondria and that Rad51p binds directly to mitochondrial DNA. In addition, a mitochondrially-targeted restriction endonuclease (mtLS-KpnI) was used to produce a unique double-strand break (DSB) in the mitochondrial genome, which allowed direct analysis of DSB repair in vivo in Saccharomyces cerevisiae. We find that loss of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs.

A new method to efficiently induce a site‐specific double‐strand break in the fission yeast Schizosaccharomyces pombe

Double-strand DNA breaks are a serious threat to cellular viability and yeast systems have proved invaluable in helping to understand how these potentially toxic lesions are sensed and repaired. An important method to study the processing of DNA breaks in the budding yeast Saccharomyces cerevisiae is to introduce a unique double-strand break into the genome by regulating the expression of the site-specific HO endonuclease with a galactose inducible promoter. Variations of the HO site-specific DSB assay have been adapted to many organisms, but the methodology has seen only limited use in the fission yeast Schizosaccharomyces pombe because of the lack of a promoter capable of inducing endonuclease expression on a relatively short time scale (~1 h). We have overcome this limitation by developing a new assay in which expression of the homing endonuclease I-PpoI is tightly regulated with a tetracycline-inducible promoter. We show that induction of the I-PpoI endonuclease produces rapid cutting of a defined cleavage site (> 80% after 1 h), efficient cell cycle arrest and significant accumulation of the checkpoint protein Crb2 at break-adjacent regions in a manner that is analogous to published findings with DSBs produced by an acute exposure to ionizing irradiation. This assay provides an important new tool for the fission yeast community and, because many aspects of mammalian chromatin organization have been well-conserved in Sz. pombe but not in S. cerevisiae, also offers an attractive system to decipher the role of chromatin structure in modulating the repair of double-stranded DNA breaks.

Abstract 1664: A functional study of missense variants localized in the DNA binding domain of BRCA2

Abstract Genetic evidence of pathogenicity is not available for most missense mutations (Variants of Uncertain Significance (VUS)) detected in the DNA binding domain (DBD) of BRCA2 by clinical genetic testing. In the absence of this evidence, functional assays may be useful for classification of the clinical relevance of these VUS. We used a cell based Homology-Directed repair reporter assay to characterize 14 known pathogenic and 18 known non-pathogenic BRCA2 missense mutations and 34 VUS from the DBD of BRCA2. We transfected each full-length wildtype and mutant BRCA2 construct into V-C8 BRCA2 deficient cells along with an I-Sce1-expression plasmid, which induces a unique double strand break in an integrated DR-GFP reporter plasmid. The protein expression levels were evaluated by immunoprecipitation and Western blotting. The ability of each BRCA2 mutant to repair the induced break was evaluated by fluorescent activated cell sorting (FACS) for GFP. The HDR assay exhibited 100% (95% CI: 75.3% - 100%) sensitivity and 100% (95% CI: 81.5% - 100%) specificity for pathogenic missense mutations. Next, we developed a statistical classifier for prediction of the pathogenicity or non-pathogenicity of BRCA2 variants based on results from the HDR assay. Using known pathogenic and non-pathogenic variants as standards we computed the probability that each variant is pathogenic or non-pathogenic based on HDR activity. A probability of 99% in favor of pathogenicity correctly classified the known pathogenic mutations and the non-pathogenic variants. The evaluation of an additional 34 variants identified 18 variants as deleterious and eight as neutral. In summary the HR assay serves as a reliable high-quality surrogate for classification of VUS located in the BRCA2 DBD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1664. doi:1538-7445.AM2012-1664

Abstract 5591: A homologous recombination assay for classifying BRCA2 missense variants

Abstract Germline mutations in BRCA2 predispose to breast, ovarian and other cancers. Although most mutations in the gene are known to be pathogenic, there is an increasing number of missense variants for which the contribution to cancer risk is unclear. These missense substitutions are referred to as VUS (variants of uncertain significance). Because loss of BRCA2 HR function leads to defective DNA double-strand break repair and an increased risk of cancer, we evaluated the utility of a cell-based Homologous Recombination (HR) assay for determination of the cancer relevance of BRCA2 VUS. Specifically, we studied VUS localized in the BRCA2 DNA binding domain (DBD) for effects on HR. We transfected full length wild-type and mutant BRCA2 constructs into V-C8 BRCA2 deficient cells along with an I-Sce1-expressing plasmid which induces an unique double strand break in an integrated DR-GFP reporter plasmid. We evaluated the ability of each BRCA2 mutant to repair the induced break by quantifying the GFP positive cells, generated by HR repair of the DNA break, with fluorescent activated cell sorting (FACS). To establish the sensitivity and specificity of the HR assay we evaluated a series of known deleterious and known neutral with the HR assay. These mutations of known effect were identified using a multifactorial likelihood approach, based on available family data, with a prior probability approach based on sequence conservation (A-GVGD) algorithm (http://agvgd.iarc.fr/agvgd_input.php). We calculated the total odds of pathogenicity for co-segregation of variants with the disease in affected carrier families, co-occurrence of VUS (in trans) with deleterious mutations and for family history of cancer of VUS and combined this odds with the A-GVGD based prior probabilities to generate the posterior probability of pathogenicity for each variant. Using this approach we successfully classified 31 VUS as either neutral or deleterious. By comparing these data with results from the HR assay we established that the assay is 100% sensitive (95% CI: 75.3% – 100%) and 100% specific (95% CI: 82.4% – 100%) for deleterious mutations in the BRCA2 DBD domain. In summary we evaluated the sensitivity and specificity of the HR assay for VUS in the DBD relative to a probability model based on family data and A-GVGD algorithm and we showed that the assay was highly sensitive and specific. Based on these results we propose that the HR assay can be used as a classifier of the clinical relevance of the VUS in the absence of direct genetic and family data. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5591. doi:10.1158/1538-7445.AM2011-5591

The Dance of Chromosomes during DNA Repair

DNA repair is an essential process for preserving genome integrity. Among the various forms of DNA damage, double-strand breaks (DSBs) are the most cytotoxic and genotoxic. To repair them, eukaryotic organisms use homologous recombination (HR): it consists of exchanging DNA strands between the broken DNA and an intact homologous DNA and it is choreographed by multi-protein complexes (1). During HR, the search for an intact homologous sequence among the whole genome is the most enigmatic stage (2). How can two homologous needles find each other in the genomic haystack? Is search the result of diffusion and chance encounters, or is there a search apparatus dedicated to bringing the homologous sequences together? To explore the choreography of the DNA and the recombination proteins during homology search, we developed an in vivo 3-colors assay in diploid yeast cells where 2 homologous chromosomes are fluorescently marked at the same locus (with GFP-Lac and RFP-Tet arrays), as well as recombination factors (CFP-tagged proteins). Using deconvolution microscopy, we tracked the movement of the two chromosomes in 3-dimensions in the absence and in the presence of a unique DSB induced near one of the marked chromosome. In the absence of DSB, we found that homologous chromosomes undergo a constrain Brownian motion with a diffusion coefficient of 4.10−4 μm2/s inside a small region of 300 nm. When a DSB is induced, the two homologous DNA become highly dynamic and homologous pairing occurs within one hour. This work is the first attempt to visualize simultaneously the movement of two homologous sequences in vivo into and out of repair centres. 1. Lisby, M., Barlow, J.H., Burgess, R.C. and Rothstein R. Cell: 118, 699-713, 2004. 2. Barzel, A. and Kupiec, M. Nature: 9, 27-37, 2007.

Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S-phase checkpoint

The Rad52 pathway has a central function in the recombinational repair of chromosome breaks and in the recovery from replication stress. Tolerance to replication stress also depends on the Mec1 kinase, which activates the DNA replication checkpoint in an Mrc1-dependent manner in response to fork arrest. Although the Mec1 and Rad52 pathways are initiated by the same single-strand DNA (ssDNA) intermediate, their interplay at stalled forks remains largely unexplored. Here, we show that the replication checkpoint suppresses the formation of Rad52 foci in an Mrc1-dependent manner and prevents homologous recombination (HR) at chromosome breaks induced by the HO endonuclease. This repression operates at least in part by impeding resection of DNA ends, which is essential to generate 3' ssDNA tails, the primary substrate of HR. Interestingly, we also observed that the Mec1 pathway does not prevent recombination at stalled forks, presumably because they already contain ssDNA. Taken together, these data indicate that the DNA replication checkpoint suppresses genomic instability in S phase by blocking recombination at chromosome breaks and permitting helpful recombination at stalled forks.

A Glycine-Arginine Domain in Control of the Human MRE11 DNA Repair Protein

Human MRE11 is a key enzyme in DNA double-strand break repair and genome stability. Human MRE11 bears a glycine-arginine-rich (GAR) motif that is conserved among multicellular eukaryotic species. We investigated how this motif influences MRE11 function. Human MRE11 alone or a complex of MRE11, RAD50, and NBS1 (MRN) was methylated in insect cells, suggesting that this modification is conserved during evolution. We demonstrate that PRMT1 interacts with MRE11 but not with the MRN complex, suggesting that MRE11 arginine methylation occurs prior to the binding of NBS1 and RAD50. Moreover, the first six methylated arginines are essential for the regulation of MRE11 DNA binding and nuclease activity. The inhibition of arginine methylation leads to a reduction in MRE11 and RAD51 focus formation on a unique double-strand break in vivo. Furthermore, the MRE11-methylated GAR domain is sufficient for its targeting to DNA damage foci and colocalization with gamma-H2AX. These studies highlight an important role for the GAR domain in regulating MRE11 function at the biochemical and cellular levels during DNA double-strand break repair.

Interplay between human DNA repair proteins at a unique double-strand break in vivo

DNA repair by homologous recombination is essential for preserving genomic integrity. The RAD51 paralogs (RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) play important roles in this process. In this study, we show that human RAD51 interacts with RAD51C-XRCC3 or RAD51B-C-D-XRCC2. In addition to being critical for RAD51 focus formation, RAD51C localizes to DNA damage sites. Inhibition of RAD51C results in a decrease in cellular proliferation consistent with a role in repairing double-strand breaks (DSBs) that occur naturally. To monitor a single DNA repair event, we developed immunofluorescence and chromatin immunoprecipitation (ChIP) methods on human cells where a unique DSB can be created in vivo. Using this system, we observed a single focus of RAD51C, RAD51 and 53BP1, which colocalized with gamma-H2AX. ChIPs revealed that endogenous human RAD51, RAD51C, RAD51D, XRCC2, XRCC3 and MRE11 proteins are recruited in the S-G2 phase of the cell cycle, while Ku80 is recruited during G1. We propose that RAD51C ensures a tight regulation of RAD51 assembly during DSB repair and plays a direct role in repairing DSBs in vivo.

Ku-dependent and Ku-independent end-joining pathways lead to chromosomal rearrangements during double-strand break repair in Saccharomyces cerevisiae.

Chromosomal double-strand breaks (DSBs) can be repaired by either homology-dependent or homology-independent pathways. Nonhomologous repair mechanisms have been relatively less well studied, despite their potential importance in generating chromosomal rearrangements. We have developed a Saccharomyces cerevisiae-based assay to identify and characterize homology-independent chromosomal rearrangements associated with repair of a unique DSB generated within an engineered URA3 gene. Approximately 1% of successfully repaired cells have accompanying chromosomal rearrangements consisting of large insertions, deletions, aberrant gene conversions, or other more complex changes. We have analyzed rearrangements in isogenic wild-type, rad52, yku80, and rad52 yku80 strains, to determine the types of events that occur in the presence or absence of these key repair proteins. Deletions were found in all strain backgrounds, but insertions were dependent upon the presence of Yku80p. A rare RAD52- and YKU80-independent form of deletion was present in all strains. These events were characterized by long one-sided deletions (up to 13 kb) and extensive imperfect overlapping sequences (7-22 bp) at the junctions. Our results demonstrate that the frequency and types of repair events depend on the specific genetic context. This approach can be applied to a number of problems associated with chromosome stability.

A novel selection system for chromosome translocations in Saccharomyces cerevisiae.

Chromosomal translocations are common genetic abnormalities found in both leukemias and solid tumors. While much has been learned about the effects of specific translocations on cell proliferation, much less is known about what causes these chromosome rearrangements. This article describes the development and use of a system that genetically selects for rare translocation events using the yeast Saccharomyces cerevisiae. A translocation YAC was created that contains the breakpoint cluster region from the human MLL gene, a gene frequently involved in translocations in leukemia patients, flanked by positive and negative selection markers. A translocation between the YAC and a yeast chromosome, whose breakpoint falls within the MLL DNA, physically separates the markers and forms the basis for the selection. When RAD52 is deleted, essentially all of the selected and screened cells contain simple translocations. The detectable translocation rates are the same in haploids and diploids, although the mechanisms involved and true translocation rates may be distinct. A unique double-strand break induced within the MLL sequences increases the number of detectable translocation events 100- to 1000-fold. This novel system provides a tractable assay for answering basic mechanistic questions about the development of chromosomal translocations.

Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome.

A simple and efficient gene replacement method, based on the recombination and repair activities of the cell, was developed. The method permits the targeted construction of markerless deletions, insertions and point mutations in the Escherichia coli chromosome. A suicide plasmid, carrying the mutant allele and the recognition site of meganuclease I- Sce I, is inserted into the genome by homologous recombination between the mutant and the wild-type (wt) alleles. Resolution of this cointegrate by intramolecular recombination of the allele pair results in either a mutant or a wt chromosome which can be distinguished by allele-specific PCR screening. The resolution process is stimulated by introducing a unique double-strand break (DSB) into the chromosome at the I- Sce I site. Cleavage by the nuclease not only enhances the frequency of resolution by two to three orders of magnitude, but also selects for the resolved products. The DSB-stimulated gene replacement method can be used in recombination-proficient E.coli cells, does not require specific growth conditions, and is potentially applicable in other microorganisms. Use of the method was demonstrated by constructing a 17-bp and a 62-kb deletion in the MG1655 chromosome. Cleavage of the chromosome induces the SOS response but does not lead to an increased mutation rate.

In vivo gap repair in Drosophila: a one-way street with many destinations.

While it has long been possible to study the process of recombination in yeast and other single-celled organisms, it has been difficult to distinguish between pathways of meiotic and mitotic recombination in multicellular eukaryotes. The experimental system described here bridges the historically separated fields of Genetic Recombination and DNA Repair in Drosophila. It is now feasible to study the repair of unique double-strand breaks induced in the Drosophila genome by the excision of a P-transposable element or by cleavage at an introduced endonuclease recognition sequence. This repair can be studied in both somatic cells and mitotically dividing germ cells. The repair of these breaks occurs mainly by copying sequence from a template located anywhere in the karyoplasm, and occurs in both male and female flies. This system, which was the first of its kind in metazoan organisms, is now being used for gene targeting in Drosophila. This review summarizes results that provide new insights into the process of gap repair in Drosophila and outline some recent experiments that demonstrate the power of the gene targeting technique.

In vivo gap repair in Drosophila: a one‐way street with many destinations

While it has long been possible to study the process of recombination in yeast and other single-celled organisms, it has been difficult to distinguish between pathways of meiotic and mitotic recombination in multicellular eukaryotes. The experimental system described here bridges the historically separated fields of Genetic Recombination and DNA Repair in Drosophila. It is now feasible to study the repair of unique double-strand breaks induced in the Drosophila genome by the excision of a P-transposable element or by cleavage at an introduced endonuclease recognition sequence. This repair can be studied in both somatic cells and mitotically dividing germ cells. The repair of these breaks occurs mainly by copying sequence from a template located anywhere in the karyoplasm, and occurs in both male and female flies. This system, which was the first of its kind in metazoan organisms, is now being used for gene targeting in Drosophila. This review summarizes results that provide new insights into the process of gap repair in Drosophila and outline some recent experiments that demonstrate the power of the gene targeting technique. BioEssays 20:317-327, 1998.© 1998 John Wiley & Sons, Inc.

Structural effect of donor DNA on the initiation of recombination for double strand break repair in human nuclear extracts.

The effect of the structure of donor DNA molecules on the initiation of recombination for double strand break repair in human nuclear extracts, was investigated here. A unique double strand break was introduced into M13 duplex derivatives by digestion with restriction enzymes. After coincubation of the cleaved DNA in human nuclear extracts, with a plasmid containing M13 sequences spanning the break, double strand break repair was estimated by the plating efficiency in JM109 (RecA1) bacteria. We first confirm that a short heterologous insert (8bp) close to the break on the recipient cleaved M13 DNA inhibits recombination with circular as well as with linear donor molecules. The results indicate that, with these substrates, recombination is initiated at the level of the break, requires uninterrupted homology on both sides of the break, and is associated with a decreasing gradient of gene conversion. When the heterologous insertion is located on the plasmid donor DNA, similar results are obtained with a circular donor DNA. In contrast, with a linear donor molecule, bearing the insert, homology requirements, in the region of the break in M13 DNA, are abolished. This last result suggests that recombination could be initiated at the extremities of the linear donor DNA.

Replication of colicin E1 plasmid DNA in cell extracts. Origin and direction of replication.

The structures of molecules of colicin E1 plasmid DNA that were in the process of being replicated in vitro were examined electron microscopically. Circular molecules containing a loop of approximately 7% of the length of the molecules were the major class of replicating molecules. The location of the loop was determined by treating the molecules with restriction endonuclease EcoR1, which introduced one unique double-strand break in the colicin E1 plasmid DNA molecules. The loops had a specific location with two branch points at approximately 17 and 24% of the molecular length from the endonuclease-sensitive site. Molecules with a larger loop were observed with a preparation labeled with 5-bromodeoxyuridine and enriched for these molecules. One of the branch points in these molecules was located at approximately 17% of the molecular length from the endonuclease-sensitive site independent of the size of the loops. These results indicate that the origin of replication of the plasmid DNA is located within the small loop and the replication proceeds unidirectionally. The molecules that had completed a round of replication had the monomeric twisted circular structure.