Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

ABSTRACT Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PKCS, but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.

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MicroRNA regulation of the MRN complex impacts DNA damage, cellular senescence and angiogenic signaling

10.1101/132258 ◽

2017 ◽

Author(s):

Cristina Espinosa-Diez ◽

RaeAnna Wilson ◽

Namita Chatterjee ◽

Clayton Hudson ◽

Rebecca Ruhl ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Targeted Delivery ◽

Genotoxic Stress ◽

Telomerase Activity ◽

Loss Of Function ◽

Mrn Complex ◽

Vegf Signaling

AbstractMicroRNAs contribute to biological robustness by buffering cellular processes from external perturbations. Here we report an unexpected link between DNA damage response and angiogenic signaling that is buffered by two distinct microRNAs. We demonstrate that genotoxic stress-induced miR-494 and miR-99b inhibit the DNA repair machinery by targeting the MRE11a-RAD50-NBN (MRN) complex. Functionally, gain and loss of function experiments show that miR-494 and miR-99b affect telomerase activity, activate p21 and Rb pathways and diminish angiogenic sproutingin vitroandin vivo. Genetic and pharmacological disruption of VEGFR-2 signaling and the MRN complex reveal a surprising co-dependency of these pathways in regulating endothelial senescence and proliferation. Vascular-targeted delivery of miR-494 decreases both growth factor-induced and tumor angiogenesis in mouse models. Mechanistically, disruption of the MRN complex induced CD44, a known driver of senescence and regulator of VEGF signaling in addition to suppressing IL-13 a stimulator of VEGF signaling. Our work identifies a putative miR-facilitated mechanism by which endothelial cells can be insulated against VEGF signaling to facilitate the onset of senescence and highlight the potential of targeting DNA repair to disrupt pathological angiogenesis.

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Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615439114 ◽

2017 ◽

Vol 114 (4) ◽

pp. E466-E475 ◽

Cited By ~ 22

Author(s):

Gábor M. Harami ◽

Yeonee Seol ◽

Junghoon In ◽

Veronika Ferencziová ◽

Máté Martina ◽

...

Keyword(s):

Single Molecule ◽

Domain Architecture ◽

Dna Recombination ◽

Recq Helicase ◽

Dna Structures ◽

Recq Helicases ◽

Differential Recognition ◽

D Loop ◽

Mechanistic Basis

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show thatEscherichia coliRecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.

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Analysis of DNA breaks, DNA damage response, and apoptosis produced by high NaCl

AJP Renal Physiology ◽

10.1152/ajprenal.90424.2008 ◽

2008 ◽

Vol 295 (6) ◽

pp. F1678-F1688 ◽

Cited By ~ 18

Author(s):

Natalia I. Dmitrieva ◽

Maurice B. Burg

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Dna Damage Response ◽

Hela Cells ◽

Cell Types ◽

Dna Breaks ◽

Main Subject ◽

Mrn Complex ◽

Damage Response

We previously reported that, both in cell culture and in the renal inner medulla in vivo, elevating NaCl increased the number of DNA breaks, which persisted as long as NaCl remained high but were rapidly repaired when NaCl was lowered. Furthermore, those breaks did not induce the DNA repair protein γH2AX or cause activation of the MRN (Mre11, Rad50, Nbs1) complex. In contrast, others recently reported that high NaCl does induce γH2AX and MRN complex formation and concluded that these activities are associated with repair of the DNA (Sheen MR, Kim SW, Jung JY, Ahn JY, Rhee JG, Kwon HM, Woo SK. Am J Physiol Renal Physiol 291: F1014–F1020, 2006). The purpose of the present studies was to resolve the disparity. The important difference is that HeLa cells, which were the main subject of the later report, are much less tolerant of high NaCl than are the mIMCD3 cells, which were our main subject. mIMCD3 cells survive levels of NaCl that kill HeLa cells by apoptosis. Here we demonstrate that in both cell types raising NaCl to a level that the cells survive (higher for mIMCD3 than HeLa) increases DNA breaks without inducing γH2AX or activating the MRN complex and that the DNA breaks persist as long as NaCl remains elevated, but are rapidly repaired when it is lowered. Importantly, in both cell types, raising NaCl further to cause apoptosis activates these DNA damage response proteins and greatly fragments DNA, associated with cell death. We conclude that γH2AX induction and MRN activation in response to high NaCl are associated with apoptosis, not DNA repair.

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Induction and Utilization of an ATM Signaling Pathway by Polyomavirus

Journal of Virology ◽

10.1128/jvi.79.20.13007-13017.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 13007-13017 ◽

Cited By ~ 82

Author(s):

Jean Dahl ◽

John You ◽

Thomas L. Benjamin

Keyword(s):

Dna Repair ◽

Protein A ◽

S Phase ◽

Productive Infection ◽

Infected Cells ◽

Cellular Dna ◽

Atm Signaling Pathway ◽

Atm Signaling

ABSTRACT Progression from G1 to S is essential for polyomavirus DNA replication and depends on the interaction of large T with the retinoblastoma gene product pRb. This virus-induced replication pathway is accompanied by p53 activation resembling a DNA damage response (12). We sought to determine whether this pathway depends in part on activation of the ATM (ataxia telangiectasia mutated) kinase and whether the virus gains advantages from this pathway beyond that of entry into S. We show that polyomavirus infection activates the S- and G2-phase checkpoints in primary as well as established mouse cells. Infected cells undergo a prolonged S phase compared to uninfected serum-stimulated cells and show no evidence of a G2→M transition before lytic death ensues. Infection is accompanied by increases in ATM activity in vitro and in the level of ATM-S1981-P in vivo. The incubation of infected cells with caffeine, a known ATM inhibitor, did not block entry into S but reduced the rate of viral compared to cellular DNA synthesis. Importantly, caffeine lowered the yields of viral DNA an average of 3- to 6-fold and those of infectious virus by as much as 10-fold. Virus yields were 10-fold lower in ATM −/− p53−/− than in ATM+/+ p53−/− mouse embryo fibroblasts, indicating a p53-independent role of ATM in productive infection. Replacement of the normal SMC1 (structural maintenance of chromosomes, or cohesin) protein, a critical ATM substrate in the DNA repair pathway, with its phosphorylation mutant SMC1S957AS966A also lowered virus yields by roughly 90%. We suggest that polyomavirus activates and utilizes a component(s) of an ATM pathway of DNA repair to prolong S phase and aid its own replication.

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Reovirus-Induced Alteration in Expression of Apoptosis and DNA Repair Genes with Potential Roles in Viral Pathogenesis

Journal of Virology ◽

10.1128/jvi.77.16.8934-8947.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8934-8947 ◽

Cited By ~ 26

Author(s):

Roberta L. DeBiasi ◽

Penny Clarke ◽

Suzanne Meintzer ◽

Robert Jotte ◽

B. K. Kleinschmidt-Demasters ◽

...

Keyword(s):

Gene Expression ◽

Dna Repair ◽

Tissue Injury ◽

Hek293 Cells ◽

Cellular Mechanisms ◽

Transcriptional Alterations ◽

Altered Gene ◽

Induced Apoptosis

ABSTRACT Reoviruses are a leading model for understanding cellular mechanisms of virus-induced apoptosis. Reoviruses induce apoptosis in multiple cell lines in vitro, and apoptosis plays a key role in virus-induced tissue injury of the heart and brain in vivo. The activation of transcription factors NF-κB and c-Jun are key events in reovirus-induced apoptosis, indicating that new gene expression is critical to this process. We used high-density oligonucleotide microarrays to analyze cellular transcriptional alterations in HEK293 cells after infection with reovirus strain T3A (i.e., apoptosis inducing) compared to infection with reovirus strain T1L (i.e., minimally apoptosis inducing) and uninfected cells. These strains also differ dramatically in their potential to induce apoptotic injury in hearts of infected mice in vivo—T3A is myocarditic, whereas T1L is not. Using high-throughput microarray analysis of over 12,000 genes, we identified differential expression of a defined subset of genes involved in apoptosis and DNA repair after reovirus infection. This provides the first comparative analysis of altered gene expression after infection with viruses of differing apoptotic phenotypes and provides insight into pathogenic mechanisms of virus-induced disease.

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Physical and Functional Association of the MRN Complex with Human Telomerase in Multiple Myeloma.

Blood ◽

10.1182/blood.v108.11.5076.5076 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5076-5076

Author(s):

James J. Driscoll ◽

Robert C. Bertheau ◽

Masood A. Shammas ◽

Rao H. Prabhala ◽

Kenneth C. Anderson ◽

...

Keyword(s):

Multiple Myeloma ◽

Dna Repair ◽

Homologous Recombination ◽

Cell Lines ◽

Catalytic Subunit ◽

Exonuclease Activity ◽

Mrn Complex ◽

Human Telomerase

Abstract Telomerase is a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes, is repressed in normal human somatic cells but is activated during development and upon malignant transformation. There is abundant evidence that the regulation of telomerase is multifactorial in mammalian cells, involving telomerase gene expression, post-translational protein-protein interactions, and protein phosphorylation. Initial experiments indicated that the catalytic subunit(s) of human telomerase (hTERT) is highly elevated in Multiple Myeloma (MM) cell lines and MM patient samples relative to normal donor plasma cells. We then exploited this highly detectable level of hTERT to address the potential role of proteins that may physically associate with the telomerase complex in MM. To this end, we investigated whether the MRN complex, a trimeric structure that consists of Mre11, RAD50 and Nbs that is evolutionarily conserved and functions in DNA repair and homologous recombination physically associates with the human telomerase complex. The MRN complex is required in vivo for a 5′ to 3′ exonuclease activity that mediates DNA recombination at double-strand breaks (DSBs). We have observed that each of the three MRN components was associated with the catalytic subunit of human telomerase (hTERT) in MM cell lines as demonstrated by in vivo co-immunoprecipitation with an hTERT monoclonal antibody under non-denaturing conditions. In addition, polyclonal antibodies to each MRN component individually immunoprecipitated the hTERT catalytic subunit. We also detected that the telomerase complex accessory proteins TRF-1 and TRF-2 are also associated with hTERT and the MRN components. TRF-2 may function as bridge coupling hTERT to the MRN complex. The association of the MRN components with hTERT was increased following treatment of myeloma cells with DNA-damaging agents indicating a functional relationship between telomere maintenance and the DNA repair pathway. The functional role of this complex is being addressed by measuring telomerase activity, telomere length and homologous recombination activity. Based upon the data that abolition of exonuclease activity in MRN mutants resulted in shortened telomeric DNA tracts, we hypothesize that this elevated MRN complex expression and its interaction with hTERT in myeloma, provides the ability to maintain telomeres and may be an important therapeutic target in MM.

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SETMAR functions in illegitimate DNA recombination and non-homologous end joining

10.1101/465138 ◽

2018 ◽

Author(s):

Michael Tellier ◽

Ronald Chalmers

Keyword(s):

Dna Repair ◽

Cell Lines ◽

Regulation Of Gene Expression ◽

Dna Recombination ◽

Nuclease Activity ◽

Wild Type ◽

Cellular Functions ◽

Dna Integration ◽

Methyltransferase Gene

AbstractIn anthropoid primates, SETMAR is a fusion between a methyltransferase gene and a domesticated DNA transposase. SETMAR has been found to be involved in several cellular functions including regulation of gene expression, DNA integration and DNA repair. These functions are thought to be mediated through the histone methyltransferase, the DNA binding and the nuclease activities of SETMAR. To better understand the cellular roles of SETMAR, we generated several U2OS cell lines expressing either wild type SETMAR or a truncated or mutated variant. We tested these cell lines with in vivo plasmid-based assays to determine the relevance of the different domains and activities of SETMAR in DNA integration and repair. We found that expressing the SET and MAR domains, but not wild type SETMAR, partially affect DNA integration and repair. The methyltransferase activity of SETMAR is also needed for an efficient DNA repair whereas we did not observe any requirement for the putative nuclease activity of SETMAR. Overall, our data support a non-essential function for SETMAR in DNA integration and repair.

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