Mechanism Of Homologous Recombination Research Articles

Template switching during DNA replication is a prevalent source of adaptive gene amplification.

Copy number variants (CNVs)-gains and losses of genomic sequences-are an important source of genetic variation underlying rapid adaptation and genome evolution. However, despite their central role in evolution little is known about the factors that contribute to the structure, size, formation rate, and fitness effects of adaptive CNVs. Local genomic sequences are likely to be an important determinant of these properties. Whereas it is known that point mutation rates vary with genomic location and local DNA sequence features, the role of genome architecture in the formation, selection, and the resulting evolutionary dynamics of CNVs is poorly understood. Previously, we have found that the GAP1 gene in Saccharomyces cerevisiae undergoes frequent and repeated amplification and selection under long-term experimental evolution in glutamine-limiting conditions. The GAP1 gene has a unique genomic architecture consisting of two flanking long terminal repeats (LTRs) and a proximate origin of DNA replication (autonomously replicating sequence, ARS), which are likely to promote rapid GAP1 CNV formation. To test the role of these genomic elements on CNV-mediated adaptive evolution, we performed experimental evolution in glutamine-limited chemostats using engineered strains lacking either the adjacent LTRs, ARS, or all elements. Using a CNV reporter system and neural network simulation-based inference (nnSBI) we quantified the formation rate and fitness effect of CNVs for each strain. We find that although GAP1 CNVs repeatedly form and sweep to high frequency in strains with modified genome architecture, removal of local DNA elements significantly impacts the rate and fitness effect of CNVs and the rate of adaptation. We performed genome sequence analysis to define the molecular mechanisms of CNV formation for 177 CNV lineages. We find that across all four strain backgrounds, between 26% and 80% of all GAP1 CNVs are mediated by Origin Dependent Inverted Repeat Amplification (ODIRA) which results from template switching between the leading and lagging strand during DNA synthesis. In the absence of the local ARS, a distal ARS can mediate CNV formation via ODIRA. In the absence of local LTRs, homologous recombination mechanisms still mediate gene amplification following de novo insertion of retrotransposon elements at the locus. Our study demonstrates the remarkable plasticity of the genome and reveals that template switching during DNA replication is a frequent source of adaptive CNVs.

Packaging of alphavirus-based self-amplifying mRNA yields replication-competent virus through a mechanism of aberrant homologous RNA recombination.

Messenger (m)RNA has taken center stage in vaccine development, gene therapy, and cancer immunotherapy. A next-generation of mRNA is the self-amplifying (sa)mRNA, which induces broad and long-lasting immunity at a lower dose which provides better clinical outcomes in conjunction with fewer adverse effects. SamRNA, also known as "replicon" RNA, encodes the replication machinery of an alphavirus together with an antigen. Efficient delivery of replicon RNA to target tissues can be accomplished by packaging the replicon RNA in virus-like replicon particles (VRPs) via co-transfection of producer cells with defective helper RNA(s) encoding the alphavirus structural proteins. During the manufacture of VRPs, however, there is a potential risk of RNA recombination, which may lead to the formation of replication-competent virus (RCV). To investigate the factors influencing the unwanted RCV formation, we evaluated how sequence homology orchestrates alphavirus RNA recombination. Several combinations of complementing alphavirus replicon and helper RNAs varying in length of sequences overlap were co-transfected in mammalian cells. The culture fluid was serially passaged to detect RCV. Nanopore sequencing of cells after the first passage in combination with amplicon-based Sanger sequencing of RCV in the culture fluid after four passages led to the detection of RNA recombination. RCV was generated between replicon and helper RNAs with sequence homology in either the non-structural or structural genes, whereas RNAs without overlapping gene regions did not generate RCV. Remarkably, no sequence overlap was detected at the recombination junction sites in the RCV genome, suggesting a mechanism of "aberrant homologous RNA recombination." Accordingly, we conclude that the alphavirus RNA recombination process leading to the formation of RCV is homology-assisted and can be prevented by avoiding sequence homology between replicon and helper RNAs.IMPORTANCEThere is a growing interest in the use of self-amplifying (sa)mRNA vectors for next-generation vaccine development, gene therapy, and cancer immunotherapy. The delivery of samRNA in the form of virus-like replicon particles (VRPs) enables efficient delivery of samRNA to target tissue. The production of these VRPs, however, suffers from contamination with replication-competent virus (RCV) that is thought to arise from recombination events between samRNA and helper RNAs for VRP packaging. The presence of RCV in samRNA in the clinical product is undesirable as alphaviruses may cause serious disease in humans. However, the underlying recombination mechanism leading to RCV is currently unknown. In our work, we demonstrate a detailed evaluation of the recombination sites, which indicates that RCV is formed through an unusual mechanism of "aberrant homologous RNA recombination." The results are useful for researchers in the field of RNA vaccine manufacture and delivery.

Remodeling the Homologous Recombination Mechanism of Yarrowia lipolytica for High-Level Biosynthesis of Squalene.

Squalene is a high-value antioxidant with many commercial applications. The use of microbial cell factories to produce squalene as an alternative to plant and animal extracts could meet increasing market demand. Yarrowia lipolytica is an excellent host for squalene production due to its high levels of acetyl-CoA and a hydrophobic environment. However, the need for precise and complicated gene editing has hindered the industrialization of this strain. Herein, the rapid construction of a strain with high squalene production was achieved by enhancing the homologous recombination efficiency in Y. lipolytica. First, remodeling of the homologous recombination efficiency resulted in a 10-fold increase in the homologous recombination rate. Next, the whole mevalonate pathway was integrated into the chromosome to enhance squalene production. Then, a higher level of squalene accumulation was achieved by increasing the level of acetyl coenzyme A and regulating the downstream steroid synthesis pathway. Finally, the squalene production reached 35 g/L after optimizing the fermentation conditions and performing a fed-batch culture in a 5 L jar fermenter. This is the highest squalene production ever reported to date by de novo biosynthesis without adding any inhibitors, paving a new path toward the industrial production of squalene and its downstream products.

The role of molecular diagnostics in the choice of therapy for biliary tract cancers

The aim of the study was to assess the frequency and clinical significance of various molecular genetic aberrations in biliary tract tumors and to determine the optimal methods of their testing. Material and Methods. We searched the literature sources containing information on predictive molecular markers relevant for the choice of therapy in biliary tract tumors in PubMed and eLibrary databases for the period from 2010 to 2023. data from 60 studies were included in this review. Results. Biliary tract tumors are characterized by poor prognosis and low sensitivity to major systemic therapies. Nevertheless, the emergence of new targeting drugs and prescription of therapy based on the results of molecular genetic analysis can increase the life expectancy and improve the quality of life of a significant proportion of patients. The most frequently detected clinically significant abnormalities in all biliary tract tumors include HER2 gene amplification/hyperexpression (5–20 % of cases), microsatellite instability (1–2 % of cases), BRAF V600E oncogene mutation (1–2 % of cases) and KRAS G12C oncogene mutation (about 1 % of cases). Specific targetable abnormalities unique to intrahepatic cholangiocarcinomas include aberrations in the gene encoding fibroblast growth factor receptor 2, FGFR2 (10–20 % of cases) and mutations in the gene encoding the enzyme isocitrate dehydrogenase 1, IDH1 (5–30 % of cases). Very rare clinically significant molecular markers for biliary tract tumors include translocations involving the receptor tyrosine kinase genes NTRK1-3, RET, ALK and ROS1. Mutations in the genes of the dNA double-strand break repair system by the mechanism of homologous recombination are also potentially significant for the choice of therapy. First of all, these are BRCA1/2 genes, hereditary mutations in which, according to two studies, are characteristic of 5–7 % of patients with biliary cancer. Although a significant part of the above-mentioned disorders can be detected by traditional molecular biological approaches such as PCR, IHC, FISH and Sanger sequencing, a comprehensive analysis of all molecular markers of predictive value in biliary tract tumors is difficult to perform without the help of next-generation sequencing (NGS). Conclusion. To improve treatment outcomes of patients with advanced and metastatic biliary tract cancer by individualizing drug therapy, it is necessary to perform comprehensive molecular genetic analysis of tumour tissue.

Overlap extension cloning of PCR products into a Gateway-compatible plasmid vector.

A PCR cloning method that combines a dual selection pGATE-1 plasmid vector and an improved overlap extension cloningwasdeveloped. This efficient andcost-effective method allows for the introduction of DNA fragments into the Gateway cloning pipeline. The cloning efficiency is facilitated by a dual selection that includes the ccdB gene and gentamicin resistance. For users of the Gateway cloning system, substantial cost saving comes from eliminating BP recombination and ligation reactions to introduce DNA fragments into pDONR or pENTR vectors. Beyond the Gateway technology, this recombination-based cloning system can be used to efficiently clone PCR amplicons by adding 24-basepair adaptor sequences that are utilized by bacterial homologous recombination mechanism.

Application of Cloning-Free Genome Engineering to Escherichia coli.

The propagation of foreign DNA in Escherichia coli is central to molecular biology. Recent advances have dramatically expanded the ability to engineer (bacterial) cells; however, most of these techniques remain time-consuming. The aim of the present work was to explore the possibility to use the cloning-free genome editing (CFGE) approach, proposed by Döhlemann and coworkers (2016), for E. coli genetics, and to deepen the knowledge about the homologous recombination mechanism. The E. coli auxotrophic mutant strains FB182 (hisF892) and FB181 (hisI903) were transformed with the circularized wild-type E. coli (i) hisF gene and hisF gene fragments of decreasing length, and (ii) hisIE gene, respectively. His+ clones were selected based on their ability to grow in the absence of histidine, and their hisF/hisIE gene sequences were characterized. CFGE method allowed the recombination of wild-type his genes (or fragments of them) within the mutated chromosomal copy, with a different recombination frequency based on the fragment length, and the generation of clones with a variable number of in tandem his genes copies. Data obtained pave the way to further evolutionary studies concerning the homologous recombination mechanism and the fate of in tandem duplicated genes.

From clinical to molecular diagnosis: relevance of diagnostic strategy in two cases of branchio-oto-renal syndrome – case report

BackgroundBranchio-oto-renal syndrome (BOR) is an autosomal dominant disorder characterized by deafness, branchiogenic malformations and renal abnormalities. Pathogenic variants in EYA1, SIX1 and SIX5 genes cause almost half of cases; copy number variants (CNV) and complex genomic rearrangements have been revealed in about 20% of patients, but they are not routinely and commonly included in the diagnostic work-up.Case presentationWe report two unrelated patients with BOR syndrome clinical features, negative sequencing for BOR genes and the identification of a 2.65 Mb 8q13.2–13.3 microdeletion.ConclusionsWe highlight the value of CNV analyses in high level of suspicion for BOR syndrome but negative sequencing for BOR genes and we propose an innovative diagnostic flow-chart to increase current detection rate. Our report confirms a mechanism of non-allelic homologous recombination as causing this recurrent 8q13.2–13.3 microdeletion. Moreover, considering the role of PRDM14 and NCOA2 genes, both involved in regulation of fertility and deleted in our patients, we suggest the necessity of a longer follow-up to monitor fertility issues or additional clinical findings.

Analysis of archaic human haplotypes suggests that 5hmC acts as an epigenetic guide for NCO recombination

BackgroundNon-crossover (NCO) refers to a mechanism of homologous recombination in which short tracks of DNA are copied between homologue chromatids. The allelic changes are typically restricted to one or few SNPs, which potentially allow for the gradual adaptation and maturation of haplotypes. It is assumed to be a stochastic process but the analysis of archaic and modern human haplotypes revealed a striking variability in local NCO recombination rates.MethodsNCO recombination rates of 1.9 million archaic SNPs shared with Denisovan hominids were defined by a linkage study and correlated with functional and genomic annotations as well as ChIP-Seq data from modern humans.ResultsWe detected a strong correlation between NCO recombination rates and the function of the respective region: low NCO rates were evident in introns and quiescent intergenic regions but high rates in splice sites, exons, 5′- and 3′-UTRs, as well as CpG islands. Correlations with ChIP-Seq data from ENCODE and other public sources further identified epigenetic modifications that associated directly with these recombination events. A particularly strong association was observed for 5-hydroxymethylcytosine marks (5hmC), which were enriched in virtually all of the functional regions associated with elevated NCO rates, including CpG islands and ‘poised’ bivalent regions.ConclusionOur results suggest that 5hmC marks may guide the NCO machinery specifically towards functionally relevant regions and, as an intermediate of oxidative demethylation, may open a pathway for environmental influence by specifically targeting recently opened gene loci.

LncRNA CTBP1-DT-encoded microprotein DDUP sustains DNA damage response signalling to trigger dual DNA repair mechanisms.

Sustaining DNA damage response (DDR) signalling via retention of DDR factors at damaged sites is important for transmitting damage-sensing and repair signals. Herein, we found that DNA damage provoked the association of ribosomes with IRES region in lncRNA CTBP1-DT, which overcame the negative effect of upstream open reading frames (uORFs), and elicited the novel microprotein DNA damage-upregulated protein (DDUP) translation via a cap-independent translation mechanism. Activated ATR kinase-mediated phosphorylation of DDUP induced a drastic ‘dense-to-loose’ conformational change, which sustained the RAD18/RAD51C and RAD18/PCNA complex at damaged sites and initiated RAD51C-mediated homologous recombination and PCNA-mediated post-replication repair mechanisms. Importantly, treatment with ATR inhibitor abolished the effect of DDUP on chromatin retention of RAD51C and PCNA, thereby leading to hypersensitivity of cancer cells to DNA-damaging chemotherapeutics. Taken together, our results uncover a plausible mechanism underlying the DDR sustaining and might represent an attractive therapeutic strategy in improvement of DNA damage-based anticancer therapies.

Stimulation of adaptive gene amplification by origin firing under replication fork constraint.

Adaptive mutations can cause drug resistance in cancers and pathogens, and increase the tolerance of agricultural pests and diseases to chemical treatment. When and how adaptive mutations form is often hard to discern, but we have shown that adaptive copy number amplification of the copper resistance gene CUP1 occurs in response to environmental copper due to CUP1 transcriptional activation. Here we dissect the mechanism by which CUP1 transcription in budding yeast stimulates copy number variation (CNV). We show that transcriptionally stimulated CNV requires TREX-2 and Mediator, such that cells lacking TREX-2 or Mediator respond normally to copper but cannot acquire increased resistance. Mediator and TREX-2 can cause replication stress by tethering transcribed loci to nuclear pores, a process known as gene gating, and transcription at the CUP1 locus causes a TREX-2-dependent accumulation of replication forks indicative of replication fork stalling. TREX-2-dependent CUP1 gene amplification occurs by a Rad52 and Rad51-mediated homologous recombination mechanism that is enhanced by histone H3K56 acetylation and repressed by Pol32 and Pif1. CUP1 amplification is also critically dependent on late-firing replication origins present in the CUP1 repeats, and mutations that remove or inactivate these origins strongly suppress the acquisition of copper resistance. We propose that replicative stress imposed by nuclear pore association causes replication bubbles from these origins to collapse soon after activation, leaving a tract of H3K56-acetylated chromatin that promotes secondary recombination events during elongation after replication fork re-start events. The capacity for inefficient replication origins to promote copy number variation renders certain genomic regions more fragile than others, and therefore more likely to undergo adaptive evolution through de novo gene amplification.

Role of PARP in TNBC: Mechanism of Inhibition, Clinical Applications, and Resistance.

Triple-negative breast cancer is a combative cancer type with a highly inflated histological grade that leads to poor theragnostic value. Gene, protein, and receptor-specific targets have shown effective clinical outcomes in patients with TNBC. Cells are frequently exposed to DNA-damaging agents. DNA damage is repaired by multiple pathways; accumulations of mutations occur due to damage to one or more pathways and lead to alterations in normal cellular mechanisms, which lead to development of tumors. Advances in target-specific cancer therapies have shown significant momentum; most treatment options cause off-target toxicity and side effects on healthy tissues. PARP (poly(ADP-ribose) polymerase) is a major protein and is involved in DNA repair pathways, base excision repair (BER) mechanisms, homologous recombination (HR), and nonhomologous end-joining (NEJ) deficiency-based repair mechanisms. DNA damage repair deficits cause an increased risk of tumor formation. Inhibitors of PARP favorably kill cancer cells in BRCA-mutations. For a few years, PARPi has shown promising activity as a chemotherapeutic agent in BRCA1- or BRCA2-associated breast cancers, and in combination with chemotherapy in triple-negative breast cancer. This review covers the current results of clinical trials testing and future directions for the field of PARP inhibitor development.

Abstract 2039: The role of HORMAD1 in DNA damage repair in squamous cell carcinomas

Abstract Genomic instability is a prominent hallmark of cancer, however the dynamic mechanisms that drive and sustain genomic instability remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability express meiosis genes and undergo a process called meiomitosis. Cancer meiomitosis is the orchestrated activation of both mitotic and meiotic machineries in neoplastic cells that confer a selective advantage. Specifically, meiCT (meiosis-specific cancer/testis) genes that specialize in the reductional division of germ cells in meiosis I are ectopically expressed in several cancers, particularly in squamous cell carcinomas. The meiCT gene, HORMAD1 is a meiosis specific protein that functions to ensure that a sufficient number of DSBs are formed for appropriate meiotic progression and to maintain genome integrity throughout homologous recombination. Studies show that HORMAD1 is significantly upregulated in several cancers and is regarded as a potentially important oncogene that plays a role in sustaining increased genomic instability. With the use of shRNA mediated knockdown of HORMAD1, we evaluated the effects ofHORMAD1 on genomic instability and survival in squamous cell carcinomas (SCCs). Our results demonstrate that HORMAD1 knockdown in SCC cancer cell lines leads to an increase in genomic instability denoted by an increase in double strand breaks, micronuclei formation and ploidy. Furthermore, shHORMAD1 results in increased sensitivity to etoposide treatment leading to increased centrosome formation and reduced cell survival. We also demonstrate thatHORMAD1 modulates a functional and balanced level of genomic instability by eliciting an DNA damage response through homologous recombination mechanisms that allows SCCs to thrive following DNA damage. Citation Format: Jennifer Gantchev, Amelia Martinez Villarreal, Brandon Ramchatesingh, Ivan V. Litvinov. The role of HORMAD1 in DNA damage repair in squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2039.

RNase H1C collaborates with ssDNA binding proteins WHY1/3 and recombinase RecA1 to fulfill the DNA damage repair in Arabidopsis chloroplasts.

Proper repair of damaged DNA is crucial for genetic integrity and organismal survival. As semi-autonomous organelles, plastids have their own genomes whose integrity must be preserved. Several factors have been shown to participate in plastid DNA damage repair; however, the underlying mechanism remains unclear. Here, we elucidate a mechanism of homologous recombination (HR) repair in chloroplasts that involves R-loops. We find that the recombinase RecA1 forms filaments in chloroplasts during HR repair, but aggregates as puncta when RNA:DNA hybrids accumulate. ssDNA-binding proteins WHY1/3 and chloroplast RNase H1 AtRNH1C are recruited to the same genomic sites to promote HR repair. Depletion of AtRNH1C or WHY1/3 significantly suppresses the binding of RNA polymerase to the damaged DNA, thus reducing HR repair and modulating microhomology-mediated double-strand break repair. Furthermore, we show that DNA polymerase IB works with AtRNH1C genetically to complete the DNA damage repair process. This study reveals the positive role of R-loops in facilitating the activities of WHY1/3 and RecA1, which in turn secures HR repair and organellar development.

Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor

Von Willebrand disease (VWD), the most common inherited bleeding disorder in humans, is caused by quantitative or qualitative defects in von Willebrand factor (VWF). VWD represents a potential target for gene therapy applications, as a single treatment could potentially result in a long-term correction of the disease. In recent years, several liver-directed gene therapy approaches have been exploited for VWD, but their efficacy was generally limited by the large size of the VWF transgene and the reduced hemostatic activity of the protein produced from hepatocytes. In this context, we aimed at developing a gene therapy strategy for gene delivery into endothelial cells, the natural site of biosynthesis of VWF. We optimized an endothelial-specific dual hybrid AAV vector, in which the large VWF cDNA was put under the control of an endothelial promoter and correctly reconstituted upon cell transduction by a combination of trans-splicing and homologous recombination mechanisms. In addition, we modified the AAV vector capsid by introducing an endothelial-targeting peptide to improve the efficiency for endothelial-directed gene transfer. This vector platform allowed the reconstitution of full-length VWF transgene both in vitro in human umbilical vein endothelial cells and in vivo in VWD mice, resulting in long-term expression of VWF.

BRCA-guided therapy of ovarian cancer

Advanced (FIGO stages III and IV) epithelial ovarian cancer (aEOC) accounts for the majority of deaths from gynecological cancers in western countries. Although the prognosis of this disease has been considerably improved in the last two decades, the majority of women will still die from progression of EOC. Optimal cytoreductive surgery and cytotoxic chemotherapy remains the mainstay of treatment in the front-line setting. Approximately 18% of EOCs harbor germline mutations of the tumor suppressor genes Breast Cancer Susceptibility Gene 1 (BRCA1) and 2 (BRCA2), while another 3–6% of these tumors have somatic mutations of these genes. These mutations lead to increased predisposition of multiple cancers. In addition, BRCA1 and BRCA2 genes encode proteins that are implicated in the Homologous Recombination (HR) mechanism, which is responsible for the repair of DNA Double Strand Breaks (DSBs), which is a common mechanism of action of chemotherapy. The incorporation of BRCA-targeted therapies, such as poly ADP ribose polymerase (PARP) inhibitors in the treatment algorithm of advanced EOC has further improved outcomes and represents a successful strategy of individualization of treatment in EOC. In this review, we summarize current treatment recommendations for patients with EOC and a BRCA1/2 mutation.

Structural insights into the inhibition of bacterial RecA by naphthalene polysulfonated compounds.

SummaryAs a promising target for alternative antimicrobials, bacterial recombinase A (RecA) protein has attracted much attention for its roles in antibiotic-driven SOS response and mutagenesis. Naphthalene polysulfonated compounds (NPS) such as suramin have previously been explored as antibiotic adjuvants targeting RecA, although the underlying structural bases for RecA-ligand interactions remain obscure. Based on our in silico predictions and documented activity of NPS in vitro, we conclude that the analyzed NPS likely interact with Tyr103 (Y103) and other key residues in the ATPase activity center (pocket A). For validation, we generated recombinant RecA proteins (wild-type versus Y103 mutant) to determine the binding affinities for RecA protein interactions with suramin and underexamined NPS in isothermal titration calorimetry. The corresponding dissociation constants (Kd) ranged from 11.5 to 18.8 μM, and Y103 was experimentally shown to be critical to RecA-NPS interactions.

Non-recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance.

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.

Evaluating the Genomic Parameters Governing rAAV-Mediated Homologous Recombination

Recombinant adeno-associated virus (rAAV) vectors have the unique ability to promote targeted integration of transgenes via homologous recombination at specified genomic sites, reaching frequencies of 0.1%-1%. We studied genomic parameters that influence targeting efficiencies on a large scale. To do this, we generated more than 1,000 engineered, doxycycline-inducible target sites in the human HAP1 cell line and infected this polyclonal population with a library of AAV-DJ targeting vectors, with each carrying a unique barcode. The heterogeneity of barcode integration at each target site provided an assessment of targeting efficiency at that locus. We compared targeting efficiency with and without target site transcription for identical chromosomal positions. Targeting efficiency was enhanced by target site transcription, while chromatin accessibility was associated with an increased likelihood of targeting. ChromHMM chromatin states characterizing transcription and enhancers in wild-type K562 cells were also associated with increased AAV-HR efficiency with and without target site transcription, respectively. Furthermore, the amenability of a site to targeting was influenced by the endogenous transcriptional level of intersecting genes. These results define important parameters that may not only assist in designing optimal targeting vectors for genome editing, but also provide new insights into the mechanism of AAV-mediated homologous recombination.

Elucidation of DNA Repair Function of PfBlm and Potentiation of Artemisinin Action by a Small-Molecule Inhibitor of RecQ Helicase.

Artemisinin (ART)-based combination therapies are recommended as first- and second-line treatments for Plasmodium falciparum malaria. Here, we investigated the impact of the RecQ inhibitor ML216 on the repair of ART-mediated damage in the genome of P. falciparumPfBLM and PfWRN were identified as members of the RecQ helicase family in P. falciparum However, the role of these RecQ helicases in DNA double-strand break (DSB) repair in this parasite has not been explored. Here, we provide several lines of evidence to establish the involvement of PfBlm in DSB repair in P. falciparum First, we demonstrate that PfBlm interacts with two well-characterized DSB repair proteins of this parasite, namely, PfRad51 and PfalMre11. Second, we found that PfBLM expression was upregulated in response to DNA-damaging agents. Third, through yeast complementation studies, we demonstrated that PfBLM could complement the DNA damage sensitivity of a Δsgs1 mutant of Saccharomyces cerevisiae, in contrast to the helicase-dead mutant PfblmK83R Finally, we observe that the overexpression of PfBLM induces resistance to DNA-damaging agents and offers a survival advantage to the parasites. Most importantly, we found that the RecQ inhibitor ML216 inhibits the repair of DSBs and thereby renders parasites more sensitive to ART. Such synergism between ART and ML216 actions was observed for both drug-sensitive and multidrug-resistant strains of P. falciparum Taken together, these findings establish the implications of PfBlm in the Plasmodium DSB repair pathway and provide insights into the antiparasitic activity of the ART-ML216 combination.IMPORTANCE Malaria continues to be a serious threat to humankind not only because of the morbidity and mortality associated with the disease but also due to the huge economic burden that it imparts. Resistance to all available drugs and the unavailability of an effective vaccine cry for an urgent discovery of newer drug targets. Here, we uncovered a role of the PfBlm helicase in Plasmodium DNA double-strand break repair and established that the parasitic DNA repair mechanism can be targeted to curb malaria. The small-molecule inhibitor of PfBlm tested in this study acts synergistically with two first-line malaria drugs, artemisinin (ART) and chloroquine, in both drug-sensitive and multidrug-resistant strains of P. falciparum, thus qualifying this chemical as a potential partner in ART-based combination therapy. Additionally, the identification of this new specific inhibitor of the Plasmodium homologous recombination (HR) mechanism will now allow us to investigate the role of HR in Plasmodium biology.

ASH2L drives proliferation and sensitivity to bleomycin and other genotoxins in Hodgkin\u2019s lymphoma and testicular cancer cells

It is of clinical importance to identify biomarkers predicting the efficacy of DNA damaging drugs (genotoxins) so that nonresponders are not unduly exposed to the deleterious effects of otherwise inefficient drugs. Here, we initially focused on the bleomycin genotoxin because of the limited information about the genes implicated in the sensitivity or resistance to this compound. Using a whole-genome CRISPR/Cas9 gene knockout approach, we identified ASH2L, a core component of the H3K4 methyl transferase complex, as a protein required for bleomycin sensitivity in L1236 Hodgkin lymphoma. Knocking down ASH2L in these cells and in the NT2D1 testicular cancer cell line rendered them resistant to bleomycin, etoposide, and cisplatin but did not affect their sensitivity toward ATM or ATR inhibitors. ASH2L knockdown decreased cell proliferation and facilitated DNA repair via homologous recombination and nonhomologous end-joining mechanisms. Data from the Tumor Cancer Genome Atlas indicate that patients with testicular cancer carrying alterations in the ASH2L gene are more likely to relapse than patients with unaltered ASH2L genes. The cell models we have used are derived from cancers currently treated either partially (Hodgkin’s lymphoma), or entirely (testicular cancer) with genotoxins. For such cancers, ASH2L levels could be used as a biomarker to predict the response to genotoxins. In situations where tumors are expressing low levels of ASH2L, which may allow them to resist genotoxic treatment, the use of ATR or ATM inhibitors may be more efficacious as our data indicate that ASH2L knockdown does not affect sensitivity to these inhibitors.