The repair of chromosomal double strand breaks (DSBs) is crucial in the maintenance of genomic integrity. However, the repair of DSBs can also destabilize the genome by causing mutations and chromosomal rearrangements, the driving forces for carcinogenesis and hereditary diseases. Break induced replication (BIR) is one of the DSB repair pathways that is highly prone to genetic instability1–3. BIR proceeds by invasion of one broken end into a homologous DNA sequence followed by replication that can copy hundreds of kilobasepairs of DNA from a donor molecule all the way through its telomere4,5. The resulting repaired chromosome comes at a great cost to the cell, as BIR promotes mutagenesis, loss of heterozygosity, translocations, and copy number variations, all hallmarks of carcinogenesis4–9. BIR employs the majority of known replication proteins to copy large portions of DNA, similar to S-phase replication10,11. It has thus been suggested that BIR proceeds by semiconservative replication; however, the model of a bona-fide, stable replication fork contradicts the known instabilities associated with BIR such as a 1000-fold increase in mutation rate compared to normal replication9. Here we demonstrate that the mechanism of replication during BIR is significantly different from S-phase replication, as it proceeds via an unusual bubble-like replication fork that results in conservative inheritance of the new genetic material. We provide the evidence that this atypical mode of DNA replication, dependent on Pif1 helicase, is responsible for the dramatic increase in BIR-associated mutations. We propose that the BIR-mode of synthesis presents a powerful mechanism that can initiate bursts of genetic instability in eukaryotes including humans.

Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half-crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here we demonstrate that HC formation results from the interruption of BIR caused by a damaged template, defective replisome or premature onset of mitosis. Additionally, we document that checkpoint failure promotes channeling of BIR into half-crossover-initiated instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. We postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.

Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

BackgroundIdentification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application.ResultsHere we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations.ConclusionOur streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2596-3) contains supplementary material, which is available to authorized users.

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Hematopoietic stem and progenitor cells (HSPCs) can generate a diversity of blood cells throughout the human lifespan. Although these cells have been evaluated using both sorted (Corces et al. Nat. Genet. 2016) and single cell assays (Pellin et al Nat. Commun. 2019), there remains uncertainty in the degree of heterogeneity within HSPC subpopulations and their associated differentiation trajectories. The phenotypic diversity within HSPCs needs to be better characterized in order to understand the pathogenesis of blood disorders including hematologic malignancies. To address this need, we characterized healthy bone marrow mononuclear cells (BMMCs) with whole transcriptome analysis (WTA) and surface marker evaluation. We hypothesized that by utilizing concurrent RNA and surface marker analysis (n=35), we can improve HSPC clustering and characterize specific phenotypic states along unique hematopoietic differentiation trajectories. Three healthy BMMC samples were stained with antibody conjugated oligonucleotides (BD® Abseq) and analyzed using BD Rhapsody™. We filtered 8,070 high quality cells for 2,508 HSPCs, myeloid cells, and lymphocyte precursors. The antibody-derived tags (ADTs) obtained from BD® Abseq comprised 33 of the most informative features (n=2000) and resulted in more stable clustering as determined by within sum of squares (WSS = 898 verses 934 for WTA alone for 20 clusters). Additionally, we designed a targeted HSPC panel (n=500 genes) with BD® Abseq which identified similar cell clusters compared to the WTA alone and WTA plus ADT data (rand index = 0.88). HSPC clustering identified putative hematopoietic stem cell (HSC), common myeloid progenitor (CMP), and megakaryocyte-erythroid progenitor (MEP) clusters that expressed canonical surface markers. Of interest, we identified candidate novel myeloid, T-cell, and B-cell primed precursors and new surface marker expression gradients that align with specific differentiation trajectories. The results of this analysis will be presented. The HSPC clusters were converted into a signature matrix using Cibersortx (Newman et al. Nat. Biotechnol. 2019), and bulk acute myeloid leukemia (AML) and healthy samples were deconvolved into respective healthy cell clusters. We subsequently performed multivariate Cox proportional hazard analysis, and observed that high levels of healthy cluster 8 (H8; HR 3.40, 95% confidence interval 1.24-9.34), the candidate lymphoid-primed multipotent progenitor (LMPP), and low levels of healthy cluster 6 (H6; HR 0.26, 95% confidence interval 0.12-0.58), a candidate erythrocyte precursor, at diagnosis were associated with worse overall survival. Deconvolution using sorted healthy sub-populations (Corces et al. Nat. Genet. 2016) only identified erythrocyte precursors as statistically relevant (HR 0.27, 95% confidence interval 0.12-0.61). Of note, H8 had a distinct gene expression profile compared to that identified for the sorted LMPP sub-population using differential gene expression analysis. In summary, we identified novel cell type clusters and surface marker associations using combined single cell WTA and surface marker analysis (BD® Abseq). We were able to correlate cell types with both canonical and novel surface markers, and deconvolution analysis provided preliminary insights into their clinical relevance in AML. Citation Format: Asiri Ediriwickrema, Sreejith Ramakrishnan, Margaret Nakamoto, Smita Ghanekar, Bogdan Luca, Aaron Newman, Andrew Gentles, Ravindra Majeti. Multiomic single cell analysis of normal human bone marrow identifies a unique stem and progenitor population that expands in AML [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 3779.