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Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-β and Ku70.

ABSTRACT: Multiple DNA repair pathways may be involved in the removal of the same DNA lesion caused by endogenous or exogenous agents. Although distinct DNA repair machinery fulfill overlapping roles in the repair of DNA lesions, the mechanisms coordinating different pathways have not been investigated in detail. Here, we show that Ku70, a core protein of nonhomologous end-joining (NHEJ) repair pathway, can directly interact with DNA polymerase-β (Pol-β), a central player in the DNA base excision repair (BER), and this physical complex not only promotes the polymerase activity of Pol-β and BER efficiency but also enhances the classic NHEJ repair. Moreover, we find that DNA damages caused by methyl methanesulfonate (MMS) or etoposide promote the formation of Ku70-Pol-β complexes at the repair foci. Furthermore, suppression of endogenous Ku70 expression by small interfering RNA reduces BER efficiency and leads to higher sensitivity to MMS and accumulation of the DNA strand breaks. Similarly, Pol-β knockdown impairs total-NHEJ capacity but only has a slight influence on alternative NHEJ. These results suggest that Pol-β and Ku70 coordinate 2-way crosstalk between the BER and NHEJ pathways.-Xia, W., Ci, S., Li, M., Wang, M., Dianov, G. L., Ma, Z., Li, L., Hua, K., Alagamuthu, K. K., Qing, L., Luo, L., Edick, A. M., Liu, L., Hu, Z., He, L., Pan, F., Guo, Z. Two-way crosstalk between BER and c-NHEJ repair pathway is mediated by Pol-β and Ku70.

SUBMITTER: Xia W

PROVIDER: S-EPMC6902736 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:BackgroundHIV-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.ResultsWe 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.ConclusionsOur 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.

| S-EPMC6833283 | biostudies-literature

LRIK interacts with the Ku70-Ku80 heterodimer enhancing the efficiency of NHEJ repair.

Project description:Despite recent advances in our understanding of the function of long noncoding RNAs (lncRNAs), their roles and functions in DNA repair pathways remain poorly understood. By screening a panel of uncharacterized lncRNAs to identify those whose transcription is induced by double-strand breaks (DSBs), we identified a novel lncRNA referred to as LRIK that interacts with Ku, which enhances the ability of the Ku heterodimer to detect the presence of DSBs. Here, we show that depletion of LRIK generates significantly enhanced sensitivity to DSB-inducing agents and reduced DSB repair efficiency. In response to DSBs, LRIK enhances the recruitment of repair factors at DSB sites and facilitates γH2AX signaling. Our results demonstrate that LRIK is necessary for efficient repairing DSBs via nonhomologous end-joining pathway.

| S-EPMC7852670 | biostudies-literature

Oxidative DNA damage is concurrently repaired by base excision repair (BER) and apyrimidinic endonuclease 1 (APE1)-initiated nonhomologous end joining (NHEJ) in cortical neurons.

Project description:AIMS:Accumulating studies have suggested that base excision repair (BER) is the major repair pathway of oxidative DNA damage in neurons, and neurons are deficient in other DNA repair pathways, including nucleotide excision repair and homologous recombination repair. However, some studies have demonstrated that neurons could efficiently repair glutamate- and menadione-induced double-strand breaks (DSBs), suggesting that the DSB repair mechanisms might be implicated in neuronal health. In this study, we hypothesized that BER and nonhomologous end joining (NHEJ) work together to repair oxidative DNA damage in neurons. METHODS:Immunohistochemistry and confocal microscopy were employed to examine the colocalization of apyrimidinic endonuclease 1 (APE1), histone variant 2AX (?H2AX) and phosphorylated p53-binding protein (53BP1). APE1 inhibitor and shRNA were respectively applied to suppress APE1 activity and protein expression to determine the correlation of APE1 and DSB formation. The neutral comet assay was used to determine and quantitate the formation of DSB. RESULTS:Both ?H2AX and 53BP1 were upregulated and colocalized with APE1 in the nuclei of rat cortical neurons subjected to menadione-induced oxidative insults. Phospho53BP1 foci were efficiently abolished, but ?H2AX foci persisted following the suppression of APE1 activity. Comet assays demonstrated that the inhibition of APE1 decreased the DSB formation. CONCLUSIONS:Our results indicate that APE1 can engage the NHEJ mechanism in the repair of oxidative DNA damage in neurons. These findings provide insights into the mechanisms underlying the efficient repair of oxidative DNA damage in neurons despite the high oxidative burden.

| S-EPMC7317839 | biostudies-literature

Alternative-NHEJ is a mechanistically distinct pathway of mammalian chromosome break repair.

Project description:Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI-induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.

| S-EPMC2430616 | biostudies-literature

Pol? tumor variants decrease the efficiency and accuracy of NHEJ.

Project description:The non homologous end-joining (NHEJ) pathway of double-strand break (DSB) repair often requires DNA synthesis to fill the gaps generated upon alignment of the broken ends, a complex task performed in human cells by two specialized DNA polymerases, Pol? and Pol?. It is now well established that Pol? is the one adapted to repair DSBs with non-complementary ends, the most challenging scenario, although the structural basis and physiological implications of this adaptation are not fully understood. Here, we demonstrate that two human Pol? point mutations, G174S and R175H, previously identified in two different tumor samples and affecting two adjacent residues, limit the efficiency of accurate NHEJ by Pol? in vitro and in vivo. Moreover, we show that this limitation is the consequence of a decreased template dependency during NHEJ, which renders the error-rate of the mutants higher due to the ability of Pol? to randomly incorporate nucleotides at DSBs. These results highlight the relevance of the 8 kDa domain of Pol? for accurate and efficient NHEJ, but also its contribution to the error-prone behavior of Pol? at 2-nt gaps. This work provides the first demonstration that mutations affecting Pol? identified in tumors can alter the efficiency and fidelity of NHEJ.

| S-EPMC5622330 | biostudies-literature

The helicase domain of Pol? counteracts RPA to promote alt-NHEJ.

Project description:Mammalian polymerase theta (Pol?) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Pol?-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Pol?-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Pol? and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.

| S-EPMC6047744 | biostudies-literature

Pol β associated complex and base excision repair factors in mouse fibroblasts.

Project description:During mammalian base excision repair (BER) of lesion-containing DNA, it is proposed that toxic strand-break intermediates generated throughout the pathway are sequestered and passed from one step to the next until repair is complete. This stepwise process is termed substrate channeling. A working model evaluated here is that a complex of BER factors may facilitate the BER process. FLAG-tagged DNA polymerase (pol) β was expressed in mouse fibroblasts carrying a deletion in the endogenous pol β gene, and the cell extract was subjected to an 'affinity-capture' procedure using anti-FLAG antibody. The pol β affinity-capture fraction (ACF) was found to contain several BER factors including polymerase-1, X-ray cross-complementing factor1-DNA ligase III and enzymes involved in processing 3'-blocked ends of BER intermediates, e.g. polynucleotide kinase and tyrosyl-DNA phosphodiesterase 1. In contrast, DNA glycosylases, apurinic/aprymidinic endonuclease 1 and flap endonuclease 1 and several other factors involved in BER were not present. Some of the BER factors in the pol β ACF were in a multi-protein complex as observed by sucrose gradient centrifugation. The pol β ACF was capable of substrate channeling for steps in vitro BER and was proficient in in vitro repair of substrates mimicking a 3'-blocked topoisomerase I covalent intermediate or an oxidative stress-induced 3'-blocked intermediate.

| S-EPMC3526277 | biostudies-literature

Oxidized nucleotide insertion by pol β confounds ligation during base excision repair.

Project description:Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase β (pol β) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol β insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol β expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides.

| S-EPMC5228075 | biostudies-literature

PARP1-MGMT complex underpins pathway crosstalk in O6-methylguanine repair

Project description:DNA lesions induced by alkylating agents are repaired by two canonical mechanisms, base excision repair dependent on poly(ADP) ribose polymerase 1 (PARP1) and the other mediated by O6-methylguanine (O6meG)-DNA methyltransferase (MGMT) in a single-step catalysis of alkyl-group removal. O6meG is the most cytotoxic and mutagenic lesion among the methyl adducts induced by alkylating agents. Although it can accomplish the dealkylation reaction all by itself as a single protein without associating with other repair proteins, evidence is accumulating that MGMT can form complexes with repair proteins and is highly regulated by a variety of post-translational modifications, such as phosphorylation, ubiquitination, and others. Here, we show that PARP1 and MGMT proteins interact directly in a non-catalytic manner, that MGMT is subject to PARylation by PARP1 after DNA damage, and that the O6meG repair is enhanced upon MGMT PARylation. We provide the first evidence for the direct DNA-independent PARP1-MGMT interaction. Further, PARP1 and MGMT proteins also interact via PARylation of MGMT leading to formation of a novel DNA damage inducible PARP1-MGMT protein complex. This catalytic interaction activates O6meG repair underpinning the functional crosstalk between base excision and MGMT-mediated DNA repair mechanisms. Furthermore, clinically relevant ‘chronic’ temozolomide exposure induced PARylation of MGMT and increased binding of PARP1 and MGMT to chromatin in cells. Thus, we provide the first mechanistic description of physical interaction between PARP1 and MGMT and their functional cooperation through PARylation for activation of O6meG repair. Hence, the PARP1-MGMT protein complex could be targeted for the development of advanced and more effective cancer therapeutics, particularly for cancers sensitive to PARP1 and MGMT inhibition. Supplementary Information The online version contains supplementary material available at 10.1186/s13045-022-01367-4.

| S-EPMC9563463 | biostudies-literature

Inactivation of Pol ? and C-NHEJ eliminates off-target integration of exogenous DNA.

Project description:Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase ? (Pol ?) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase ? and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase ? and classical non-homologous recombination eliminates random integration.

| S-EPMC5501794 | biostudies-literature

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