Browse
Submit Data
Databases
API
Help

Dataset Information

4 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Structural evidence for an in trans base selection mechanism involving Loop1 in polymerase μ at an NHEJ double-strand break junction.

ABSTRACT: Eukaryotic DNA polymerase (Pol) X family members such as Pol μ and terminal deoxynucleotidyl transferase (TdT) are important components for the nonhomologous DNA end-joining (NHEJ) pathway. TdT participates in a specialized version of NHEJ, V(D)J recombination. It has primarily nontemplated polymerase activity but can take instructions across strands from the downstream dsDNA, and both activities are highly dependent on a structural element called Loop1. However, it is unclear whether Pol μ follows the same mechanism, because the structure of its Loop1 is disordered in available structures. Here, we used a chimeric TdT harboring Loop1 of Pol μ that recapitulated the functional properties of Pol μ in ligation experiments. We solved three crystal structures of this TdT chimera bound to several DNA substrates at 1.96-2.55 Å resolutions, including a full DNA double-strand break (DSB) synapsis. We then modeled the full Pol μ sequence in the context of one these complexes. The atomic structure of an NHEJ junction with a Pol X construct that mimics Pol μ in a reconstituted system explained the distinctive properties of Pol μ compared with TdT. The structure suggested a mechanism of base selection relying on Loop1 and taking instructions via the in trans templating base independently of the primer strand. We conclude that our atomic-level structural observations represent a paradigm shift for the mechanism of base selection in the Pol X family of DNA polymerases.

SUBMITTER: Loc'h J

PROVIDER: S-EPMC6615693 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Molecular basis for DNA double-strand break annealing and primer extension by an NHEJ DNA polymerase.

Project description:Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways. The mechanisms by which breaks are competently brought together and extended during NHEJ is poorly understood. As polymerases extend DNA in a 5'-3' direction by nucleotide addition to a primer, it is unclear how NHEJ polymerases fill in break termini containing 3' overhangs that lack a primer strand. Here, we describe, at the molecular level, how prokaryotic NHEJ polymerases configure a primer-template substrate by annealing the 3' overhanging strands from opposing breaks, forming a gapped intermediate that can be extended in trans. We identify structural elements that facilitate docking of the 3' ends in the active sites of adjacent polymerases and reveal how the termini act as primers for extension of the annealed break, thus explaining how such DSBs are extended in trans. This study clarifies how polymerases couple break-synapsis to catalysis, providing a molecular mechanism to explain how primer extension is achieved on DNA breaks.

| S-EPMC3898472 | biostudies-literature

Altered hematopoiesis in mice lacking DNA polymerase mu is due to inefficient double-strand break repair.

Project description:Polymerase micro (Polmicro) is an error-prone, DNA-directed DNA polymerase that participates in non-homologous end-joining (NHEJ) repair. In vivo, Polmicro deficiency results in impaired Vkappa-Jkappa recombination and altered somatic hypermutation and centroblast development. In Polmicro(-/-) mice, hematopoietic development was defective in several peripheral and bone marrow (BM) cell populations, with about a 40% decrease in BM cell number that affected several hematopoietic lineages. Hematopoietic progenitors were reduced both in number and in expansion potential. The observed phenotype correlates with a reduced efficiency in DNA double-strand break (DSB) repair in hematopoietic tissue. Whole-body gamma-irradiation revealed that Polmicro also plays a role in DSB repair in non-hematopoietic tissues. Our results show that Polmicro function is required for physiological hematopoietic development with an important role in maintaining early progenitor cell homeostasis and genetic stability in hematopoietic and non-hematopoietic tissues.

| S-EPMC2638008 | biostudies-literature

DNA methylation is stable after MMEJ and NHEJ double strand break repair

Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.

2022-09-11 | E-MTAB-12037 | biostudies-arrayexpress

Metastasis Suppressor NME1 Modulates Choice of Double-Strand Break Repair Pathways in Melanoma Cells by Enhancing Alternative NHEJ while Inhibiting NHEJ and HR.

Project description:Reduced NME1 expression in melanoma cell lines, mouse models of melanoma, and melanoma specimens in human patients is associated with increased metastatic activity. Herein, we investigate the role of NME1 in repair of double-stranded breaks (DSBs) and choice of double-strand break repair (DSBR) pathways in melanoma cells. Using chromatin immunoprecipitation, NME1 was shown to be recruited rapidly and directly to DSBs generated by the homing endonuclease I-PpoI. NME1 was recruited to DSBs within 30 min, in concert with recruitment of ataxia-telangiectasia mutated (ATM) protein, an early step in DSBR complex formation, as well as loss of histone 2B. NME1 was detected up to 5 kb from the break site after DSB induction, suggesting a role in extending chromatin reorganization away from the repair site. shRNA-mediated silencing of NME1 expression led to increases in the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways of double-strand break repair (DSBR), and reduction in the low fidelity, alternative-NHEJ (A-NHEJ) pathway. These findings suggest low expression of NME1 drives DSBR towards higher fidelity pathways, conferring enhanced genomic stability necessary for rapid and error-free proliferation in invasive and metastatic cells. The novel mechanism highlighted in the current study appears likely to impact metastatic potential and therapy-resistance in advanced melanoma and other cancers.

| S-EPMC7460576 | biostudies-literature

DNA polymerase θ (POLQ), double-strand break repair, and cancer.

Project description:DNA polymerase theta (pol θ) is encoded in the genomes of many eukaryotes, though not in fungi. Pol θ is encoded by the POLQ gene in mammalian cells. The C-terminal third of the protein is a family A DNA polymerase with additional insertion elements relative to prokaryotic homologs. The N-terminal third is a helicase-like domain with DNA-dependent ATPase activity. Pol θ is important in the repair of genomic double-strand breaks (DSBs) from many sources. These include breaks formed by ionizing radiation and topoisomerase inhibitors, breaks arising at stalled DNA replication forks, breaks introduced during diversification steps of the mammalian immune system, and DSB induced by CRISPR-Cas9. Pol θ participates in a route of DSB repair termed "alternative end-joining" (altEJ). AltEJ is independent of the DNA binding Ku protein complex and requires DNA end resection. Pol θ is able to mediate joining of two resected 3' ends harboring DNA sequence microhomology. "Signatures" of Pol θ action during altEJ are the frequent utilization of longer microhomologies, and the insertion of additional sequences at joining sites. The mechanism of end-joining employs the ability of Pol θ to tightly grasp a 3' terminus through unique contacts in the active site, allowing extension from minimally paired primers. Pol θ is involved in controlling the frequency of chromosome translocations and preserves genome integrity by limiting large deletions. It may also play a backup role in DNA base excision repair. POLQ is a member of a cluster of similarly upregulated genes that are strongly correlated with poor clinical outcome for breast cancer, ovarian cancer and other cancer types. Inhibition of pol θ is a compelling approach for combination therapy of radiosensitization.

| S-EPMC5114520 | biostudies-literature

DNA Methylation is stable after MMEJ and NHEJ double strand break repair - OCI-AML3 dataset

2022-08-25 | E-MTAB-12061 | biostudies-arrayexpress

Polymerase δ promotes chromosomal rearrangements and imprecise double-strand break repair.

Project description:Recent studies have implicated DNA polymerases θ (Pol θ) and β (Pol β) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.

| S-EPMC7959522 | biostudies-literature

Polymerase μ in non-homologous DNA end joining: importance of the order of arrival at a double-strand break in a purified system.

Project description:During non-homologous DNA end joining (NHEJ), bringing two broken dsDNA ends into proximity is an essential prerequisite for ligation by XRCC4:Ligase IV (X4L4). This physical juxtaposition of DNA ends is called NHEJ synapsis. In addition to the key NHEJ synapsis proteins, Ku, X4L4, and XLF, it has been suggested that DNA polymerase mu (pol μ) may also align two dsDNA ends into close proximity for synthesis. Here, we directly observe the NHEJ synapsis by pol μ using a single molecule FRET (smFRET) assay where we can measure the duration of the synapsis. The results show that pol μ alone can mediate efficient NHEJ synapsis of 3' overhangs that have at least 1 nt microhomology. The abundant Ku protein in cells limits the accessibility of pol μ to DNA ends with overhangs. But X4L4 can largely reverse the Ku inhibition, perhaps by pushing the Ku inward to expose the overhang for NHEJ synapsis. Based on these studies, the mechanistic flexibility known to exist at other steps of NHEJ is now also apparent for the NHEJ synapsis step.

| S-EPMC7144918 | biostudies-literature

NHEJ-deficient DT40 cells have increased levels of immunoglobulin gene conversion: evidence for a double strand break intermediate.

Project description:Activation-induced cytidine deaminase (AID) likely initiates immunoglobulin gene-conversion (GC) by deaminating cytidines within the V-region of chicken B-cells. However, the intervening DNA lesion required to initiate GC remains elusive. GC could be initiated by a single strand break or a double strand break (DSB). To distinguish between these possibilities, we examined GC in the chicken DT40 B cell line deficient in non-homologous end joining (NHEJ). It is known that the NHEJ and homologous recombination DNA repair pathways compete for DSBs. In light of this, if a DSB is the major intermediate, deficiency in NHEJ should result in increased levels of GC. Here we show that DNA-PKcs(-/-/-) and Ku70(-/-) DT40 cells had 5- to 10-fold higher levels of GC relative to wildtype DT40 as measured by surface IgM reversion and sequencing of the V-region. These data suggest that a DSB is the major DNA lesion that initiates GC.

| S-EPMC1669771 | biostudies-literature

Mu insertions are repaired by the double-strand break repair pathway of Escherichia coli.

Project description:Mu is both a transposable element and a temperate bacteriophage. During lytic growth, it amplifies its genome by replicative transposition. During infection, it integrates into the Escherichia coli chromosome through a mechanism not requiring extensive DNA replication. In the latter pathway, the transposition intermediate is repaired by transposase-mediated resecting of the 5' flaps attached to the ends of the incoming Mu genome, followed by filling the remaining 5 bp gaps at each end of the Mu insertion. It is widely assumed that the gaps are repaired by a gap-filling host polymerase. Using the E. coli Keio Collection to screen for mutants defective in recovery of stable Mu insertions, we show in this study that the gaps are repaired by the machinery responsible for the repair of double-strand breaks in E. coli-the replication restart proteins PriA-DnaT and homologous recombination proteins RecABC. We discuss alternate models for recombinational repair of the Mu gaps.

| S-EPMC3325207 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data