Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

Schwer, Bjoern; Wei, Pei‐Chi; Chang, Anna; Kao, Jennifer; Du, Zhou; Meyers, Robin M.; Alt, Frederick W.

doi:10.1073/pnas.1525564113

Cited by 93 publications

(81 citation statements)

References 45 publications

(81 reference statements)

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“…However, thus far, LAM-HTGTS can detect all known classes of recurrent DSBs across the genome, including DSBs introduced by on- or off-target activities of antigen receptor diversification enzymes 8,9 and by on- and off-target activities of engineered nucleases 6 . The assay also detects individual DSBs that occur at lower frequency but are associated with a specific cellular process across the genome, such as active transcription start sites 11,21 and sets of DSBs spread across long gene bodies in neural stem and progenitor cells that generate fragile sites 7 . Finally, the assay also detects low-level wide-spread breaks, such as those generated by ionizing radiation 6 .…”

Section: Advantages Of Lam-htgtsmentioning

confidence: 99%

“…6,9,13 . Both methods also detect DSBs generated via endogenous sources such as transcription-associated DSBs and DSBs associated with replication stress 7,11,21 , programmed DSB-inducing activities in lymphoid cells 8-13,30 , and likely could be applied to detect endogenous DSBs that arise from other sources such as oxidative DNA damage. More generally, LAM-HTGTS reveals the various classes of DSBs across the genome that can contribute to inter- or intra-chromosomal translocations and deletions, including sources of DSBs that contribute to known oncogenic translocations 8,9 .…”

Section: Detecting Endogenous Dsb and Joining With Lam-htgtsmentioning

confidence: 99%

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Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing

et al. 2016

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Unbiased, high-throughput assays to detect and quantify DNA double-stranded breaks (DSBs) genome-wide in mammalian cells will facilitate basic studies of mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as evaluating on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for detecting genome-wide “prey” DSBs via their translocation in cultured mammalian cells to a fixed “bait” DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina paired-end Miseq sequencing. A custom bioinformatic pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis are necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable, and straightforward to implement with a turnaround time of less than one week.

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Section: Advantages Of Lam-htgtsmentioning

confidence: 99%

Section: Detecting Endogenous Dsb and Joining With Lam-htgtsmentioning

confidence: 99%

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing

et al. 2016

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“…For example, the Fos promoter is already in the transcriptionally permissive state: it is trimethylated on histone H3 Lys4 (H3K4me3) and pre-bound by Pol II and TOP2β, as well as by transcription factors including cyclic AMP-responsive element-binding protein and serum response factor, which themselves are regulated by synaptic activity. Furthermore, DSBs are present near TSSs in neural stem cells and neural progenitor cells, and recurrent DSB clusters are located within the bodies of long, transcribed, late-replicating genes 36,37 . Importantly, both TOP2β and TOP1 are essential for transcription regulation of neuronal genes that are longer than 200 kb, and their length-dependent effect on gene expression is attributed to impaired transcription elongation 38 .…”

Section: Dna Breaks During Transcriptionmentioning

confidence: 99%

Physiological functions of programmed DNA breaks in signal-induced transcription

Puc

Aggarwal

Rosenfeld

2017

Nat Rev Mol Cell Biol

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The idea that signal-dependent transcription might involve the generation of transient DNA nicks or even breaks in the regulatory regions of genes, accompanied by activation of DNA damage repair pathways, would seem to be counterintuitive, as DNA damage is usually considered harmful to cellular integrity. However, recent studies have generated a substantial body of evidence that now argues that programmed DNA single- or double-strand breaks can, at least in specific cases, have a role in transcription regulation. Here, we discuss the emerging functions of DNA breaks in the relief of DNA torsional stress and in promoter and enhancer activation.

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“…A-EJ also substantially contributes to joining other types of DSBs in core C-NHEJ-deficient cycling cells (11,12).…”

mentioning

confidence: 99%

PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line

Kumar

Frock

2016

Proc. Natl. Acad. Sci. U.S.A.

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105

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Classical nonhomologous end joining (C-NHEJ) is a major mammalian DNA double-strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation. The XRCC4-like-factor (XLF) C-NHEJ protein is dispensable for V(D)J recombination in normal cells, but because of functional redundancy, it is absolutely required for this process in cells deficient for the ataxia telangiectasia-mutated (ATM) DSB response factor. The recently identified paralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to ionizing radiation-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B-cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B-cell line, and dispensable for normal ionizing radiation resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to ionizing radiation exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested pro-B-cell lines. Finally, we also find that PAXX deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ.T o repair DNA double-strand breaks (DSBs), mammalian cells use two major DSB repair pathways: homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). HR requires a long homologous template for repair and is active in S/G2 cell cycle phase (1). In contrast, C-NHEJ, which directly ligates DSBs with short (1-4 bp) or no homologies, functions throughout interphase, including during the G1 cell cycle phase (2-4). C-NHEJ repairs diverse DSBs, including those arising ectopically by ionizing radiation as well as DSBs generated as intermediates during V(D)J recombination in developing progenitor (pro)-B and -T lymphocytes and Ig heavy chain (IgH) class switch recombination (CSR) in activated mature B lymphocytes (5). The C-NHEJ pathway has a number of characterized members, including "core" C-NHEJ factors that were so-named in part based on their requirement for joining known types of ends via C-NHEJ and their evolutionary conservation (6). Such core factors include the Ku70 and Ku80 proteins, which form the "Ku" end recognition complex, and the XRCC4 and DNA Ligase4 proteins, which form the C-NHEJ ligation complex (5, 6).Exons that encode Ig and T-cell receptor (TCR) variable region exons are assembled from germline V, D, and J gene segments. This V(D)J recombinatio...

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Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells

Cited by 93 publications

References 45 publications

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing

Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing

Physiological functions of programmed DNA breaks in signal-induced transcription

PAXX and XLF DNA repair factors are functionally redundant in joining DNA breaks in a G1-arrested progenitor B-cell line

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