scholarly journals NHEJ pathway is involved in post-integrational DNA repair due to Ku70 binding to HIV-1 integrase

Retrovirology ◽  
2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Ekaterina Knyazhanskaya ◽  
Andrey Anisenko ◽  
Olga Shadrina ◽  
Anastasia Kalinina ◽  
Timofei Zatsepin ◽  
...  
Keyword(s):  
Dna Repair ◽  
Wild Type Virus ◽  
Dna Breaks ◽  
End Joining ◽  
Double Stranded Dna ◽  
Repair Efficiency ◽  
Non Homologous End Joining ◽  
Cellular Dna ◽  
Successful Replication ◽  
Hiv 1

Abstract Background HIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process. Results We engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs. Conclusions Our data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

p53 Ser15 phosphorylation disrupts the p53–RPA70 complex and induces RPA70-mediated DNA repair in hypoxia

2012 ◽  
Vol 443 (3) ◽  
pp. 811-820 ◽  
Author(s):  
Esha Madan ◽  
Rajan Gogna ◽  
Uttam Pati
Keyword(s):  
Dna Repair ◽  
Cancer Cells ◽  
Excision Repair ◽  
Protein A ◽  
Cancer Therapeutics ◽  
End Joining ◽  
Nhej Repair ◽  
Dependent Protein ◽  
Non Homologous End Joining ◽  
Cellular Dna

Cellular stressors are known to inhibit the p53–RPA70 (replication protein A, 70 kDa subunit) complex, and RPA70 increases cellular DNA repair in cancer cells. We hypothesized that regulation of RPA70-mediated DNA repair might be responsible for the inhibition of apoptosis in hypoxic tumours. We have shown that, in cancer cells, hypoxia disrupts the p53–RPA70 complex, thereby enhancing RPA70-mediated NER (nucleotide excision repair)/NHEJ (non-homologous end-joining) repair. In normal cells, RPA70 binds to the p53-NTD (N-terminal domain), whereas this binding is disrupted in hypoxia. Phosphorylation of p53-NTD is a crucial event in dissociating both NTD–RPA70 and p53–RPA70 complexes. Serial mutations at serine and threonine residues in the NTD confirm that p53Ser15 phosphorylation induces dissociation of the p53–RPA70 complex in hypoxia. DNA-PK (DNA-dependent protein kinase) is shown to induce p53Ser15 phosphorylation, thus enhancing RPA70-mediated NER/NHEJ repair. Furthermore, RPA70 gene silencing induces significant increases in cellular apoptosis in the resistant hypoxic cancer cells. We have thus elucidated a novel pathway showing how DNA-PK-mediated p53Ser15 phosphorylation dissociates the p53–RPA70 complex, thus enhancing NER/NHEJ repair, which causes resistance to apoptosis in hypoxic cancer cells. This novel finding may open new strategies in developing cancer therapeutics on the basis of the regulation of RPA70-mediated NER/NHEJ repair.

scholarly journals Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aron Ferenczi ◽  
Yen Peng Chew ◽  
Erika Kroll ◽  
Charlotte von Koppenfels ◽  
Andrew Hudson ◽  
...  
Keyword(s):  
Dna Repair ◽  
Chlamydomonas Reinhardtii ◽  
Nuclear Dna ◽  
Model Organism ◽  
Single Strand ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Non Homologous End Joining ◽  
Underlying Mechanisms

AbstractSingle-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates in CRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strand templated DNA repair (SSTR) are inadequately understood, constraining rational improvements to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalga Chlamydomonas reinhardtii. We demonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologous recombination and Fanconi anemia pathways, is strongly antagonized by non-homologous end-joining, and is mediated almost entirely by the alternative end-joining enzyme polymerase θ. These findings suggest differences in SSTR between C. reinhardtii and animals. Our work illustrates the promising potentially of C. reinhardtii as a model organism for studying nuclear DNA repair.

scholarly journals Knock-In Reporter Mice Demonstrate that DNA Repair by Non-homologous End Joining Declines with Age

PLoS Genetics ◽  
2014 ◽  
Vol 10 (7) ◽  
pp. e1004511 ◽  
Author(s):  
Amita Vaidya ◽  
Zhiyong Mao ◽  
Xiao Tian ◽  
Brianna Spencer ◽  
Andrei Seluanov ◽  
...  
Keyword(s):  
Dna Repair ◽  
End Joining ◽  
Reporter Mice ◽  
Non Homologous End Joining

scholarly journals Double-strand DNA breaks recruit the centromeric histone CENP-A

2009 ◽  
Vol 106 (37) ◽  
pp. 15762-15767 ◽  
Author(s):  
Samantha G. Zeitlin ◽  
Norman M. Baker ◽  
Brian R. Chapados ◽  
Evi Soutoglou ◽  
Jean Y. J. Wang ◽  
...  
Keyword(s):  
Dna Cleavage ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Double Strand Dna Breaks ◽  
Histone H3 Variant ◽  
Radiation Induced ◽  
Non Homologous End Joining

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair.

scholarly journals Genetic Variants of ATM & Non-Homologous End Joining Pathway Genes in Acute Myeloid Leukemia: A South India Case-Control Study

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5076-5076
Author(s):  
Sugunakar Vuree ◽  
Anuradha Cingeetham ◽  
Dunna Nageswara Rao ◽  
Manjula Gorre ◽  
Sudha Sinha ◽  
...  
Keyword(s):  
Dna Repair ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Case Control Study ◽  
Case Control ◽  
South Indian Population ◽  
End Joining ◽  
Non Homologous End Joining ◽  
Control Study ◽  
Acute Myeloid

Purpose of the study: Deregulated DNA repair is one of the hallmarks of cancers including Acute Myeloid Leukemia (AML), as it results in genomic instability. ATM gene functions as a sensor, activates cascade of events leading to stimulation of multiple DNA damage- responsive signaling pathways. Principal DNA repair mechanism activated in the hematopoietic stem cells is the Non Homologous End Joining (NHEJ) pathway. However, this pathway was shown to be error prone. Functional SNPs in the genes involved in DNA repair might influence the gene expression leading to altered DNA repair which might confer the risk to AML. Materials & Methods: This hospital-based case-control study included 225 AML patients and 326 cancer-free controls from South Indian population. Six polymorphisms of XRCC5, XRCC6, XRCC7 and ATM were genotyped using polymerase chain reaction (PCR)-Restriction Fragment Length Polymorphism (PCR- RFLP) method. Statistical analyses were performed by using SPSS (version20v) and SNPSTAT online tool. Protein-Protein Interaction (PPI) analysis was also done to see the relationship between these genes. Results: We found that there was an elevated risk of AML associated with the XRCC5 VNTR 0R repeat and A allele of 2408G>A polymorphism (p-0.04 and p<0.0001 respectively), the frequencies of G allele (p-<0.0001) of XRCC6 -1310C>G and T allele (p-0.003) of ATM -5144A>T polymorphisms were also significantly increased in AML cases. Further, analyses of the variant genotypes with epidemiological and clinical variables revealed a significant association of the risk genotypes with development and progression of AML. Conclusion: The XRCC5 0R repeat, 2408G>A, XRCC6 -1310 C>G and ATM- 5144A>T polymorphisms, but not XRCC6 -61C>G and XRCC7 6721G>T polymorphisms, play an important role in the pathogenesis of AML. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Increased Sensitivity of PBMCs Isolated from Patients with Rheumatoid Arthritis to DNA Damaging Agents Is Connected with Inefficient DNA Repair

2020 ◽  
Vol 9 (4) ◽  
pp. 988
Author(s):  
Grzegorz Galita ◽  
Olga Brzezińska ◽  
Izabela Gulbas ◽  
Joanna Sarnik ◽  
Marta Poplawska ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Dna Repair ◽  
Mononuclear Cells ◽  
Excision Repair ◽  
Dna Lesions ◽  
Dna Breaks ◽  
Peripheral Blood Mononuclear ◽  
Dna Damaging Agents ◽  
Repair Efficiency ◽  
Systemic Inflammatory Disease

Rheumatoid arthritis (RA) is a systemic, inflammatory disease of the joints and surrounding tissues. RA manifests itself with severe joint pain, articular inflammation, and oxidative stress. RA is associated with certain types of cancer. We have assumed that RA patients’ increased susceptibility to cancer may be linked with genomic instability induced by impaired DNA repair and sensitivity to DNA damaging agents. The aim of this work was to analyze the sensitivity of peripheral blood mononuclear cells (PBMCs) isolated from RA patients to DNA damaging agents: tert-butyl hydroperoxide (TBH), bleomycin, ultraviolet (UV) radiation, and methyl methanesulfonate (MMS) and calculate the repair efficiency. TBH induce oxidative DNA lesions repaired mainly by base excision repair (BER). Bleomycin induced mainly DNA double-strand breaks repaired by non-homologous end joining (NHEJ) and homologous recombination repair (HRR). We included 20 rheumatoid arthritis patients and 20 healthy controls and used an alkaline version of the comet assay with modification to measure sensitivity to DNA damaging agents and DNA repair efficiency. We found an increased number of DNA breaks and alkali-labile sites in the RA patients compared to those in the controls. Exposure to DNA damaging agents evoked the same increased damage in both groups, but we observed statistically higher PMBC sensitivity to TBH, MMS, bleomycin as well as UV. Examination of the repair kinetics of both groups revealed that the DNA lesions induced by TBH and bleomycin were more efficiently repaired in the controls than in the patients. These data suggest impaired DNA repair in RA patients, which may accelerate PBMC aging and/or lead to higher cancer incidence among RA patients.

Abstract 2827: Alternative non-homologous end joining DNA repair as therapeutic target in multiple myeloma

2018 ◽  
Author(s):  
Daniele Caracciolo ◽  
Martina Montesano ◽  
Emanuela Altomare ◽  
Grazia Consolo ◽  
Nicola Amodio ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dna Repair ◽  
Therapeutic Target ◽  
End Joining ◽  
Non Homologous End Joining
Sign in / Sign up

Export Citation Format

Share Document