scholarly journals Babesia bovis Rad51 ortholog influences switching of ves genes but is not essential for segmental gene conversion in antigenic variation

2020 ◽  
Author(s):  
Erin A. Mack ◽  
Massimiliano S. Tagliamonte ◽  
Yu-Ping Xiao ◽  
Samantha Quesada ◽  
David R. Allred
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Gene Conversion ◽  
Genome Stability ◽  
Antigenic Variation ◽  
Babesia Bovis ◽  
Γ Irradiation ◽  
Exogenous Dna

AbstractThe tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This makes BbRad51 a choice target for development of inhibitors that could both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here a failure to upregulate the Bbrad51 gene in response to DNA damage. Moreover, we demonstrate that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.Author summaryB. bovis establishes highly persistent infections in cattle, in part by using cytoadhesion to avoid passage through the spleen. While protective, a host antibody response targeting the cytoadhesion ligand is quickly rendered ineffective by antigenic variation. In B. bovis, antigenic variation relies heavily upon segmental gene conversion (SGC), presumed to be a form of homologous recombination (HR), to generate variants. As Rad51 is generally considered essential to HR, we investigated its contribution to SGC. While diminishing the parasite’s capacity for HR-dependent integration of exogenous DNA, the loss of BbRad51 did not affect the parasite’s sensitivity to ionizing radiation, overall genome stability, or competence for SGC. Instead, loss of BbRad51 diminished the extent of in situ transcriptional switching (isTS) among ves gene loci, the accumulation of SGC recombinants, and the mean lengths of SGC sequence tracts. Given the overall reductions in VESA1 variability, compromise of the parasite’s capacity for in vivo persistence is predicted.

scholarly journals Chromosomal double-strand breaks induce gene conversion at high frequency in mammalian cells.

1997 ◽  
Vol 17 (11) ◽  
pp. 6386-6393 ◽  
Author(s):  
D G Taghian ◽  
J A Nickoloff
Keyword(s):  
Homologous Recombination ◽  
Gene Conversion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Double Strand Breaks ◽  
Chromosomal Dna ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Conversion Tract

Double-strand breaks (DSBs) stimulate chromosomal and extrachromosomal recombination and gene targeting. Transcription also stimulates spontaneous recombination by an unknown mechanism. We used Saccharomyces cerevisiae I-SceI to stimulate recombination between neo direct repeats in Chinese hamster ovary (CHO) cell chromosomal DNA. One neo allele was controlled by the dexamethasone-inducible mouse mammary tumor virus promoter and inactivated by an insertion containing an I-SceI site at which DSBs were introduced in vivo. The other neo allele lacked a promoter but carried 12 phenotypically silent single-base mutations that create restriction sites (restriction fragment length polymorphisms). This system allowed us to generate detailed conversion tract spectra for recipient alleles transcribed at high or low levels. Transient in vivo expression of I-SceI increased homologous recombination 2,000- to 10,000-fold, yielding recombinants at frequencies as high as 1%. Strikingly, 97% of these products arose by gene conversion. Most products had short, bidirectional conversion tracts, and in all cases, donor neo alleles (i.e., those not suffering a DSB) remained unchanged, indicating that conversion was fully nonreciprocal. DSBs in exogenous DNA are usually repaired by end joining requiring little or no homology or by nonconservative homologous recombination (single-strand annealing). In contrast, we show that chromosomal DSBs are efficiently repaired via conservative homologous recombination, principally gene conversion without associated crossing over. For DSB-induced events, similar recombination frequencies and conversion tract spectra were found under conditions of low and high transcription. Thus, transcription does not further stimulate DSB-induced recombination, nor does it appear to affect the mechanism(s) by which DSBs induce gene conversion.

scholarly journals Sam68/KHDRBS1-dependent NF-κB activation confers radioprotection to the colon epithelium in γ-irradiated mice

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Kai Fu ◽  
Xin Sun ◽  
Eric M Wier ◽  
Andrea Hodgson ◽  
Ryan P Hobbs ◽  
...  
Keyword(s):  
Dna Damage ◽  
Critical Role ◽  
Genotoxic Stress ◽  
Whole Body ◽  
Colon Tissue ◽  
Γ Irradiation ◽  
Apoptotic Genes

Previously we reported that Src-associated-substrate-during-mitosis-of-68kDa (Sam68/KHDRBS1) is pivotal for DNA damage-stimulated NF-κB transactivation of anti-apoptotic genes (Fu et al., 2016). Here we show that Sam68 is critical for genotoxic stress-induced NF-κB activation in the γ-irradiated colon and animal and that Sam68-dependent NF-κB activation provides radioprotection to colon epithelium in vivo. Sam68 deletion diminishes γ-irradiation-triggered PAR synthesis and NF-κB activation in colon epithelial cells (CECs), thus hampering the expression of anti-apoptotic molecules in situ and facilitating CECs to undergo apoptosis in mice post whole-body γ-irradiation (WBIR). Sam68 knockout mice suffer more severe damage in the colon and succumb more rapidly from acute radiotoxicity than the control mice following WBIR. Our results underscore the critical role of Sam68 in orchestrating genotoxic stress-initiated NF-κB activation signaling in the colon tissue and whole animal and reveal the pathophysiological relevance of Sam68-dependent NF-κB activation in colonic cell survival and recovery from extrinsic DNA damage.

scholarly journals hSSB2 (NABP1) is required for the recruitment of RPA during the cellular response to DNA UV damage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Didier Boucher ◽  
Ruvini Kariawasam ◽  
Joshua Burgess ◽  
Adrian Gimenez ◽  
Tristan E. Ocampo ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Excision Repair ◽  
Protein A ◽  
Ultraviolet B ◽  
Repair System ◽  
Uvb Radiation ◽  
Exogenous Dna

AbstractMaintenance of genomic stability is critical to prevent diseases such as cancer. As such, eukaryotic cells have multiple pathways to efficiently detect, signal and repair DNA damage. One common form of exogenous DNA damage comes from ultraviolet B (UVB) radiation. UVB generates cyclobutane pyrimidine dimers (CPD) that must be rapidly detected and repaired to maintain the genetic code. The nucleotide excision repair (NER) pathway is the main repair system for this type of DNA damage. Here, we determined the role of the human Single-Stranded DNA Binding protein 2, hSSB2, in the response to UVB exposure. We demonstrate that hSSB2 levels increase in vitro and in vivo after UVB irradiation and that hSSB2 rapidly binds to chromatin. Depletion of hSSB2 results in significantly decreased Replication Protein A (RPA32) phosphorylation and impaired RPA32 localisation to the site of UV-induced DNA damage. Delayed recruitment of NER protein Xeroderma Pigmentosum group C (XPC) was also observed, leading to increased cellular sensitivity to UVB. Finally, hSSB2 was shown to have affinity for single-strand DNA containing a single CPD and for duplex DNA with a two-base mismatch mimicking a CPD moiety. Altogether our data demonstrate that hSSB2 is involved in the cellular response to UV exposure.

scholarly journals The role of Sp1 in the detection and elimination of cells with persistent DNA strand breaks

NAR Cancer ◽  
2020 ◽  
Vol 2 (2) ◽  
Author(s):  
Polina S Loshchenova ◽  
Svetlana V Sergeeva ◽  
Sally C Fletcher ◽  
Grigory L Dianov
Keyword(s):  
Dna Damage ◽  
Cancer Therapy ◽  
Genome Stability ◽  
Dna Strand Breaks ◽  
Dna Lesions ◽  
Exogenous Dna ◽  
Strand Breaks ◽  
Specificity Factor ◽  
Dna Strand

Abstract Maintenance of genome stability suppresses cancer and other human diseases and is critical for organism survival. Inevitably, during a life span, multiple DNA lesions can arise due to the inherent instability of DNA molecules or due to endogenous or exogenous DNA damaging factors. To avoid malignant transformation of cells with damaged DNA, multiple mechanisms have evolved to repair DNA or to detect and eradicate cells accumulating unrepaired DNA damage. In this review, we discuss recent findings on the role of Sp1 (specificity factor 1) in the detection and elimination of cells accumulating persistent DNA strand breaks. We also discuss how this mechanism may contribute to the maintenance of physiological populations of healthy cells in an organism, thus preventing cancer formation, and the possible application of these findings in cancer therapy.

scholarly journals TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

2010 ◽  
Vol 30 (12) ◽  
pp. 2971-2982 ◽  
Author(s):  
Eladio Abreu ◽  
Elena Aritonovska ◽  
Patrick Reichenbach ◽  
Gaël Cristofari ◽  
Brad Culp ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Immunoprecipitation ◽  
Shelterin Complex ◽  
Damage Response ◽  
Single Stranded Region ◽  
Ob Fold ◽  
Per Se ◽  
Human Telomerase

ABSTRACT Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase; however, the molecular basis for recruitment is not known. Here, we have directly investigated the process of telomerase recruitment via fluorescence in situ hybridization (FISH) and chromatin immunoprecipitation (ChIP). We find that depletion of two components of the shelterin complex that is found at telomeres—TPP1 and the protein that tethers TPP1 to the complex, TIN2—results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Deletion of the oligonucleotide/oligosaccharide binding fold (OB-fold) of TPP1 disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere.

scholarly journals PML Colocalizes with and Stabilizes the DNA Damage Response Protein TopBP1

2003 ◽  
Vol 23 (12) ◽  
pp. 4247-4256 ◽  
Author(s):  
Zhi-Xiang Xu ◽  
Anna Timanova-Atanasova ◽  
Rui-Xun Zhao ◽  
Kun-Sang Chang
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Genome Stability ◽  
Growth Regulation ◽  
Suppressor Gene ◽  
Nuclear Body ◽  
Promyelocytic Leukemia ◽  
Multifunctional Protein ◽  
Damage Response

ABSTRACT The PML tumor suppressor gene is consistently disrupted by t(15;17) in patients with acute promyelocytic leukemia. Promyelocytic leukemia protein (PML) is a multifunctional protein that plays essential roles in cell growth regulation, apoptosis, transcriptional regulation, and genome stability. Our study here shows that PML colocalizes and associates in vivo with the DNA damage response protein TopBP1 in response to ionizing radiation (IR). Both PML and TopBP1 colocalized with the IR-induced bromodeoxyuridine single-stranded DNA foci. PML and TopBP1 also colocalized with Rad50, Brca1, ATM, Rad9, and BLM. IR and interferon (IFN) coinduce the expression levels of both TopBP1 and PML. In PML-deficient NB4 cells, TopBP1 was unable to form IR-induced foci. All-trans-retinoic acid induced reorganization of the PML nuclear body (NB) and reappearance of the IR-induced TopBP1 foci. Inhibition of PML expression by siRNA is associated with a significant decreased in TopBP1 expression. Furthermore, PML-deficient cells express a low level of TopBP1, and its expression cannot be induced by IR or IFN. Adenovirus-mediated overexpression of PML in PML−/− mouse embryo fibroblasts substantially increased TopBP1 expression, which colocalized with the PML NBs. These studies demonstrated a mechanism of PML-dependent expression of TopBP1. PML overexpression induced TopBP1 protein but not the mRNA expression. Pulse-chase labeling analysis demonstrated that PML overexpression stabilized the TopBP1 protein, suggesting that PML plays a role in regulating the stability of TopBP1 in response to IR. Together, our findings demonstrate that PML regulates TopBP1 functions by association and stabilization of the protein in response to IR-induced DNA damage.

scholarly journals Targeting Homologous Recombination in Lymphoid Malignancies: Evaluation of Four Small Molecule Inhibitors of RAD51

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2080-2080
Author(s):  
Melinda Day ◽  
Tyler Maclay ◽  
Amber Cyr ◽  
Muneer G Hasham ◽  
Kin-hoe Chow ◽  
...  
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Cell Line ◽  
Genomic Instability ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Burkitt’S Lymphoma ◽  
Lymphoid Malignancies ◽  
Anti Tumor Activity

Genomic instability is recognized as a driver of tumorigenesis and cancer progression. Loss of tumor suppressors or activation of oncogenes can induce DNA damage stress, promoting genomic instability and creating dependencies upon key DNA repair pathways. These dependencies can be targeted therapeutically to induce synthetic lethality. The homologous recombination (HR) repair pathway is an attractive target. HR deficient cancers are hypersensitive to numerous anticancer drugs, and tumors will often induce expression of HR genes to promote drug resistance. RAD51 is a key component of the HR pathway. RAD51 forms nucleoprotein filaments at sites of DNA damage and replication fork stalls, mediating homologous DNA strand exchange to promote recombinational repair of breaks and damaged replication forks. We utilized four small molecule inhibitors of RAD51-mediated HR for evaluation of RAD51 as a potential therapeutic target. Compounds CYT-0851, CYT-0853, CYT-1027, and CYT-1127 were evaluated for anti-cancer activity in vitro and in vivo. To determine the impact of the small molecules on RAD51 and HR, all four were tested for effects on RAD51 focus formation and sister chromatid exchange (SCE) activity. All the compounds showed a reduction in SCE activity, however only CYT-0851 and CYT-0853 produced a measurable reduction in RAD51 foci. We have previously shown that that RAD51 inhibition leads to accumulation of DNA breaks, and ultimately cell death, in cells expressing the DNA mutator protein Activation Induced Cytidine deaminase (AICDA/AID). Cytotoxicity assays were performed in an AID+ (Daudi, Burkitt's Lymphoma) and AID- (WI-38, fibroblast) cell lines. All four compounds were preferentially active in AID+ cells with little to no cytotoxicity observed in the AID-negative WI-38 cell line. CYT-0853 was the most potent in the Daudi cell line with an EC50 of 8nM. All four compounds were orally bioavailable in all preclinical species tested but showed differences in pharmacokinetics. Preclinical cell line derived xenograft models of AID-high Burkitt's lymphoma (Daudi) and B-cell acute lymphoblastic leukemia (CCRF-SB) were used to determine the in vivo anti-tumor activity of the compounds in lymphoid cancer models. CYT-0851 and CYT-0853 both showed significant anti-tumor activity with tumor growth inhibition of greater than 50% in both models. Further analysis showed drug exposure with CYT-0851 was more consistent in the CDX models than CYT-0853. Overall, these data indicate that RAD51 and HR are attractive therapeutic targets for the treatment of lymphoid malignancies and that CYT-0851 is a viable clinical development candidate. Disclosures Day: Cyteir Therapeutics: Employment. Maclay:Cyteir Therapeutics: Employment. Cyr:Cyteir Therapeutics: Employment. Mills:Cyteir Therapeutics: Employment, Equity Ownership.

scholarly journals ATRIP protects progenitor cells against DNA damage in vivo

2020 ◽  
Author(s):  
Gabriel E. Matos-Rodrigues ◽  
Paulius Grigaravicius ◽  
Bernard S. Lopez ◽  
Thomas Hofmann ◽  
Pierre-Olivier Frappart ◽  
...  
Keyword(s):  
Dna Damage ◽  
Progenitor Cells ◽  
Genome Stability ◽  
Genomic Stability ◽  
Interacting Protein ◽  
Replicative Stress ◽  
Dependent Cell ◽  
Short Life Expectancy

AbstractThe maintenance of genomic stability during the cell cycle of progenitor cells is essential for the faithful transmission of genetic information. Mutations in genes that ensure genome stability lead to human developmental syndromes. Mutations in Ataxia Telangiectasia and Rad3-related (ATR) or in ATR-interacting protein (ATRIP) lead to Seckel syndrome, which is characterized by developmental malformations and short life expectancy. While the roles of ATR in replicative stress response and chromosomal segregation are well established, it is unknown how ATRIP contributes to maintaining genomic stability in progenitor cells in vivo. Here, we generated the first mouse model to investigate ATRIP function. Conditional inactivation of Atrip in progenitor cells of the CNS and eye led to microcephaly, microphthalmia and postnatal lethality. To understand the mechanisms underlying these malformations, we used lens progenitor cells as a model and found that ATRIP loss promotes replicative stress and TP53-dependent cell death. Trp53 inactivation in Atrip-deficient progenitor cells rescued apoptosis but increased mitotic DNA damage and mitotic defects. Our findings demonstrate an essential role of ATRIP in preventing DNA damage accumulation during unchallenged replication.

scholarly journals Molecular flexibility of DNA as a key determinant of RAD51 recruitment

2018 ◽  
Author(s):  
Federico Paoletti ◽  
Afaf El-Sagheer ◽  
Jun Allard ◽  
Tom Brown ◽  
Omer Dushek ◽  
...  
Keyword(s):  
Dna Damage ◽  
Surface Plasmon Resonance ◽  
Homologous Recombination ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Genome Stability ◽  
General Framework ◽  
Binding Kinetics ◽  
Double Stranded Dna ◽  
Conceptual Question

AbstractThe timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 polymerises faster on single-stranded DNA (ssDNA) compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By fitting detailed polymerisation models informed by our experimental datasets, we show that RAD51 is a mechano-sensor that exhibits a larger polymerisation rate constant on flexible ssDNA compared to rigid ssDNA or dsDNA. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in rapidly recruiting repair factors that multimerise at sites of DNA damage.

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