Neuronal enhancers are hotspots for DNA single-strand break repair
Open accessPublished version of a preprint
Abstract
Defects in DNA repair frequently lead to neurodevelopmental and neurodegenerative diseases, underscoring the particular importance of DNA repair in long-lived post-mitotic neurons 1,2. The cellular genome is subjected to a constant barrage of endogenous DNA damage, but surprisingly little is known about the identity of the lesion(s) that accumulate in neurons and whether they accrue throughout the genome or at specific loci. Here we show that post-mitotic neurons accumulate unexpectedly high levels of DNA single-strand breaks (SSBs) at specific sites within the genome. Genome-wide mapping reveals that SSBs are located within enhancers at or near CpG dinucleotides and sites of DNA demethylation. These SSBs are repaired by PARP1 and XRCC1-dependent mechanisms. Notably, deficiencies in XRCC1-dependent short-patch repair increase DNA repair synthesis at neuronal enhancers, whereas defects in long-patch repair reduce synthesis. The high levels of SSB repair in neuronal enhancers are therefore likely to be sustained by both short-patch and long-patch processes. These data provide the first evidence of site- and cell-type-specific SSB repair, revealing unexpected levels of localized and continuous DNA breakage in neurons. In addition, they suggest an explanation for the neurodegenerative phenotypes that occur in patients with defective SSB repair.
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The process of BER erasing oxidative damaged bases and cytosine methylation could lead to base mutations.
“This process promotes extensive endogenous DNA damage in neuronal enhancers, safeguards cell identities but could also lead to base mutations12–15.”
From Single-cell parallel analysis of DNA damage and transcriptome reveals selective genome vulnerability (2025) · cites this paper as [12]
In “SSBs and active cytosine demethylation” (page not provided), the paper links cytosine demethylation at neuronal enhancers to base excision repair. Its Conclusions (page not provided) say recurrent repair synthesis could increase mutations. It does not establish that repair of oxidative base damage causes the enhancer damage studied; it notes that oxidative lesions have not been shown to preferentially accumulate at enhancers. (LLM, conf=0.87)
Experimental — accuracy not yet measuredThe process of BER erasing oxidative damaged bases and cytosine methylation promotes extensive endogenous DNA damage in neuronal enhancers.
“This process promotes extensive endogenous DNA damage in neuronal enhancers, safeguards cell identities but could also lead to base mutations12–15.”
From Single-cell parallel analysis of DNA damage and transcriptome reveals selective genome vulnerability (2025) · cites this paper as [12]
The “SSBs and active cytosine demethylation” section reports elevated endogenous single-strand breaks at neuronal enhancers and identifies cytosine methylation–demethylation cycles as a potential source, not an established cause. It says oxidative lesions have not been shown to preferentially accumulate at enhancers, so it does not support attributing the enhancer damage to BER removal of oxidatively damaged bases. The Conclusions raise a possibility of mutations at recurrent repair sites but do not establish that this process safeguards cell identity. (LLM, conf=0.86)
Experimental — accuracy not yet measuredThe process of BER erasing oxidative damaged bases and cytosine methylation safeguards cell identities.
“This process promotes extensive endogenous DNA damage in neuronal enhancers, safeguards cell identities but could also lead to base mutations12–15.”
From Single-cell parallel analysis of DNA damage and transcriptome reveals selective genome vulnerability (2025) · cites this paper as [12]
The “SSBs and active cytosine demethylation” section links cytosine demethylation at neuronal enhancers to intermediates of base excision repair, while the Conclusions suggest that recurrent repair there could increase mutations. The paper does not say this process safeguards cell identities. It also says oxidative lesions have not been shown to preferentially accumulate at enhancers, so it does not establish their removal as the source of the enhancer damage described in the citing sentence. Page numbers are unavailable in our copy. (LLM, conf=0.84)
Experimental — accuracy not yet measuredDNA repair hotspots are enriched in a subset of regulatory regions including enhancers, to protect the identities and functions of postmitotic cells (such as neurons).
“Recent studies revealed that DNA repair hotspots are enriched in a subset of regulatory regions including enhancers, to protect the identities and functions of postmitotic cells (such as neurons)12,13,15.”
From Single-cell parallel analysis of DNA damage and transcriptome reveals selective genome vulnerability (2025) · cites this paper as [12]
The ‘DNA repair sites enriched at enhancers’ section reports recurrent repair synthesis at neuronal enhancers, including 90% of identified super-enhancers, which the paper describes as involved in cell identity. The Conclusions suggest that this repair may matter for neurological function, but do not establish that the hotspots protect neuronal identity or function; that purpose is stronger than the paper’s finding. (LLM, conf=0.84)
Experimental — accuracy not yet measuredThe observed DNA damage hotspots are combined results from DNA damage formation and repair.
“Moreover, the observed DNA damage hotspots are combined results from DNA damage formation and repair12,13.”
From Single-cell parallel analysis of DNA damage and transcriptome reveals selective genome vulnerability (2025) · cites this paper as [12]
Neurons actively induce both DSBs and single-stranded breaks as part of their normal physiology.
“neurons also actively induce both DSBs and single-stranded breaks as part of their normal physiology”
From Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration (2023) · cites this paper as [8]
Extensive active demethylation following neuronal activity through the Tet-TDG pathway-associated base excision repair can be a source of DNA breaks.
“extensive active demethylation following neuronal activity through the Tet-TDG pathway-associated base excision repair can also be a source of DNA breaks”
From Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration (2023) · cites this paper as [8]
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References 82 · Cited by 223
- Authors
- Wu, W., Kargbo-Hill, S. E., Nathan, W. J., Paiano, J., Callen, E., Wang, D., Shinoda, K., van Wietmarschen, N., Colón-Mercado, J. M., Zong, D., De Pace, R., Shih, H.-Y., Coon, S., Parsadanian, M., Pavani, R., Hanzlikova, H., Park, S., Jung, S. K., McHugh, P. J., Canela, A., Chen, C., Casellas, R., Caldecott, K. W., Ward, M. E., & Nussenzweig, A.
- Affiliations
- National Institutes of Health (US)
- National Institute of Neurological Disorders and Stroke (US)
- John Radcliffe Hospital (GB)
- University of Sussex (GB)
- Corresponding author
- Caldecott, Keith W.
- Ward, Michael E.
- Nussenzweig, André
- ORCID
- Venue
- Nature
- Published
- 2021-03-25
- Volume
- 593
- Issue
- 7859
- Pages
- 440-444
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- PMC
- PMC9827709
- OpenAlex
- W3139535723
- Cited by
- 223
- References
- 82
- Citations by year
- 2026: 29 · 2025: 43 · 2024: 41 · 2023: 44 · 2022: 49 · 2021: 17
- FWCI
- 11.79
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Licence
- Locations
- Nature (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Sussex Research Online (University of Sussex) (submittedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- Figshare (submittedVersion, cc-by)
- Funders
- U.S. Department of Defense (W81XWH-16-1-599, W81XWH1910652, W81XWH)
- Ellison Medical Foundation (AG-SS- 2633-11)
- Alex's Lemonade Stand Foundation for Childhood Cancer
- BrightFocus Foundation
- National Institutes of Health (MR/P010121/1, 694996, W81XWH)
- Medical Research Council (MR/P010121/1, 694996)
- Japan Society for the Promotion of Science (20K21393, 19H03208)
- National Cancer Institute (Z01BC010283)
- National Institute of Neurological Disorders and Stroke
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Health Sciences › Medicine › Oncology › PARP inhibition in cancer therapy
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Keywords
- single-strand breaks, DNA repair, postmitotic neurons, CpG, DNA demethylation, PARP1, XRCC1, short-patch repair, long-patch repair, genome-wide mapping, neurodegeneration, neurodevelopmental disorders, endogenous DNA damage
- MeSH terms
- Cell Line, DNA, DNA Repair, DNA Replication, Enhancer Elements, Genetic, Humans, Male, Methylation, Neurons, Poly(ADP-ribose) Polymerases, Sequence Analysis, DNA, 5-Methylcytosine, DNA Breaks, Single-Stranded
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