Neuronal Enhancers are Hotspots For DNA Single-Strand Break Repair

Wei Wu, Sarah E. Hill, William J. Nathan, Jacob Paiano, Kenta Shinoda, Jennifer Colon-Mercado, Elsa Callen, Raffaella de Pace, Dongpeng Wang, Han-Yu Shih, Steve Coon, Maia Parsadanian, Hana Hanzlikova, Peter J. McHugh, Andres Canela, Keith W. Caldecott, Michael E. Ward, André Nussenzweig

Preprint posted on December 16, 2020

Article now published in Nature at

Endogenous DNA breaks at neuronal enhancers: novel links to neurodegeneration?

Selected by Giuseppina D'Alessandro, Ram

Categories: cell biology


Our cells have evolved various DNA repair pathways to deal with DNA lesions that constantly challenge the stability of our genome. Defects in DNA repair may lead to the development of cancer and immunological or neurological disorders (Jackson and Bartek, 2009). Repair of various DNA lesions, including single-strand breaks (SSBs), involves a gap-filling step, which incorporates one or several nucleotides into the damaged DNA using the undamaged DNA strand as a template.

Although neurons face different kinds of DNA damaging insults, defects in the SSB repair pathway seem to impact predominantly their function (Caldecott, 2008; Tubbs A, 2017). Indeed, hereditary mutations in genes encoding for SSB repair proteins, like XRCC1, are mainly associated with neurological phenotypes.

However, the extent and genome-wide distribution of SSB repair signatures in neurons are not clear. Thus, to gauge the genome-wide distribution of DNA repair signatures in post-mitotic neurons, the authors of this preprint developed a novel technique called SAR-seq and report that neurons accumulate SSBs in enhancer regions of the genome.


Key findings

  1. SAR-seq identifies sites of unscheduled DNA synthesis

Unscheduled DNA synthesis occurs during DNA repair to fill the gaps generated by excised or missing nucleotides (also known as gap filling). To map sites of DNA repair synthesis genome-wide, the authors developed a new method: synthesis-associated with repair sequencing (SAR-seq, fig 1a). For this purpose, they labelled iPSC-derived neurons (i3 neurons) with the thymidine analogue EdU, which is incorporated at the sites of DNA synthesis. The labelled DNA was biotinylated by click chemistry, sheared, and isolated for high-throughput sequencing. They identified >55,000 peaks at recurrent genomic locations around 200-2,000 bp in width, possibly reflecting clustered DNA repair loci. SAR peaks were also observed in rat neurons but not in other post-mitotic cells like G0-arrested pre-B cells or iPSC-derived skeletal muscles. Notably, SAR peaks were unaffected by inhibitors of the replicative DNA polymerases (α, δ, and ϵ), reinforcing that i3 neurons are not replicating and the SAR signal is from unscheduled DNA synthesis.

Figure 1: (a) Schematic of SAR-seq (DNA synthesis associated with repair sequencing) methodology, (b) Heatmaps of SAR-seq, H3K4me1, H3K27ac, MLL4 ChIP-seq and ATAC-seq signal ±1kb around SAR-seq peak summits in i3Neurons, ordered by SAR-seq intensity, (c) Graph showing the fold enrichment of SAR-seq and ATAC-seq peaks located at enhancers and promoters compared to 1000 sets of randomly shuffled regions of the same sizes and chromosome distributions, respectively. Graph showing the fraction of super-enhancers overlapping with SAR-seq peaks compared to conventional enhancers, and (d) Heatmaps of XRCC1 and PAR ChIP-seq signal ±1kb surrounding SAR-seq peak summits in i3Neurons, ordered by SAR-seq intensity. Taken and modified directly from Wu W et. al., 2020 under a CC-BY 4.0 international license


  1. Neuronal enhancers are hotspots for unscheduled DNA synthesis

They found that neuronal SAR peaks were enriched in intragenic regions and associated with expressed genes. In particular, ~50% of the top-5000 peaks harboured a transcription factor motif – ONECUT – that is known to promote chromatin accessibility. Furthermore, these peaks also coincided with ATAC-seq peaks (a readout of open chromatin), suggesting that an open chromatin structure may influence the extent of DNA synthesis. Intriguingly, a majority of SAR peaks were enriched at cis-regulating enhancer elements (rather than promoters), as measured by enhancer histone modifications (H3K4me1, H3K27ac) and Lysine Methyltransferase 2B (MLL4) (ChIP-seq, fig 1b). Adding, most of the SAR peaks occupied super-enhancers that are active in differentiated neurons (fig 1c). Gene Ontology (GO) analysis also revealed that genes containing SAR peaks were related to neuronal functions (like neuronal migration, development, axon formation, and synapse assembly).

  1. Neuronal DNA synthesis coincides with SSB and PARP activation loci

DNA double-strand breaks (DSBs) induced by Topoisomerase II (TOP2) can promote the expression of early response genes in neurons (Madabhushi, 2015). However, DNA synthesis associated with TOP2-induced lesions did not overlap with the SAR peaks observed in untreated neurons (reiterating that SSBs mostly occur in untreated i3 neurons). Additionally, no DSBs were detected in untreated neurons, as monitored by immunofluorescence for DSB markers or END-seq, a technique that maps DSBs (Canela, 2016).

Furthermore, they found that SAR peaks corresponded to sites of PARylation and XRCC1 recruitment, key events in SSB repair (assayed using ChIP-seq, fig 1d). In SSB repair either a single nucleotide or longer DNA patches are replaced respectively by DNA polymerase β (short-patch SSB) or DNA polymerase δ, ϵ or l (long-patch SSB). The authors observed that PARP1, XRCC1, or DNA polymerase β depletion increased SAR peaks, suggesting reliance on long-patch repair when the short-patch repair is challenged (fig 2).

Figure 2: Model proposed by the authors. Defective short-patch SSB repair in post-mitotic neurons results in increased long-patch SSB. Taken and modified directly from Wu W et. al., 2020 under a CC-BY 4.0 international license.


Conclusion and perspective

In summary, this study reports that in human post-mitotic neurons, SSBs and unscheduled DNA synthesis occurs within enhancers. The presence of neurodegenerative phenotypes in patients with mutations in XRCC1 suggests a key role for short-patch SSB repair in neuronal viability (fig 2). The authors speculate that loss of XRCC1 function could lead to neuronal dysfunction by aberrant PARP1 activity or by the accumulation of mutations that could result in aberrant transcription and neurodegeneration.

Future work could reveal how different non-replicating cells deploy different DNA repair pathways and how endogenous DNA damage supports the normal physiology of different kinds of cells.


Acknowledgments: We are thankful to all the authors for their support and for taking the time to comment on the preLight.



  • Caldecott, K. W. Single-strand break repair and genetic disease. Nat Rev Genet (2008).
  • Canela, al., DNA Breaks and End Resection Measured Genome-wide by End Sequencing, Mol Cell (2016).
  • Jackson, S. P. & Bartek, J. The DNA-damage response in human biology and disease, Nature (2009).
  • Madabhushi, al., Activity-Induced DNA Breaks Govern the Expression of Neuronal Early-Response Genes, Cell (2015).
  • Mellén M et al., 5-hydroxymethylcytosine accumulation in postmitotic neurons results in functional demethylation of expressed genes, Proc Natl Acad Sci U S A. (2017).
  • Puc J,et al. Ligand-dependent enhancer activation regulated by topoisomerase-I activity, Cell (2015)
  • Tubbs A, & Nussenzweig A. Endogenous DNA Damage as a Source of Genomic Instability in Cancer, Cell (2017).

Tags: enhancers, neurons, parp1, single-strand breaks, xrcc1

Posted on: 6th January 2021


Read preprint (No Ratings Yet)

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka, Madhuja Samaddar, Miguel V. Almeida, Sejal Davla, Jennifer Ann Black, Dey Lab

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Preprint missing? Don't hesitate to let us know.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar, Ramona Jühlen, Amanda Haage, Laura McCormick, Maiko Kitaoka

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage