High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors
- University of New Mexico, United States
- Washington State University, United States
- Georgia State University, United States
Abstract
DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N-methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data shows that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs Abf1 and Reb1, but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro, causing slow repair both within the core motif and its adjacent DNA. Repair of UV damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.
Data availability
New DNA sequencing data has been deposited to GEO under accession code GSE183622. All data generated or analyzed are included in the manuscript and supplemental file. Source data files containing the numerical data for Figure 1 and Figure 2 are uploaded. Source codes used for sequencing reads mapping to identify alkylation lesions and repair analysis at yeast Abf1 and Reb1 binding sites are also uploaded.
-
Analysis of alkylation damage formation and base excision repair at yeast transcription factor binding sitesNCBI Gene Expression Omnibus, GSE183622.
-
CPD-seq mapping of transcription-coupled DNA repair in yeastNCBI Gene Expression Omnibus, GSE145911.
-
Genome-wide Maps of Alkylation Damage, Repair, and Mutagenesis in Yeast Reveal Mechanisms of Mutational HeterogeneityNCBI Gene Expression Omnibus, GSE98031.
-
A high-resolution protein architecture of the budding yeast genomeNCBI Gene Expression Omnibus, GSE147927.
Article and author information
Author details
Smitha Sivapragasam
Funding
National Institute of Environmental Health Sciences (R21ES029302)
- John J Wyrick
- Peng Mao
National Institute of Environmental Health Sciences (R01ES032814)
- John J Wyrick
National Institute of Environmental Health Sciences (R01ES028698)
- John J Wyrick
National Science Foundation (MCB 2028902)
- Gregory MK Poon
National Institute of General Medical Sciences (P20GM130422)
- Peng Mao
National Cancer Institute (P30CA118100)
- Mingrui Duan
- Peng Mao
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2022, Duan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 1,351
- views
-
- 191
- downloads
-
- 8
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
High-resolution mapping demonstrates inhibition of DNA excision repair by transcription factors
eLife 11:e73943.
https://doi.org/10.7554/eLife.73943
Further reading
-
- Cancer Biology
Expression of NPRL2/TUSC4, a tumor-suppressor gene, is reduced in many cancers including NSCLC. Restoration of NPRL2 induces DNA damage, apoptosis, and cell-cycle arrest. We investigated NPRL2 antitumor immune responses in aPD1R/KRAS/STK11mt NSCLC in humanized-mice. Humanized-mice were generated by transplanting fresh human cord blood-derived CD34 stem cells into sub-lethally irradiated NSG mice. Lung-metastases were developed from KRAS/STK11mt/aPD1R A549 cells and treated with NPRL2 w/wo pembrolizumab. NPRL2-treatment reduced lung metastases significantly, whereas pembrolizumab was ineffective. Antitumor effect was greater in humanized than non-humanized-mice. NPRL2 + pembrolizumab was not synergistic in KRAS/STK11mt/aPD1R tumors but was synergistic in KRASwt/aPD1S H1299. NPRL2 also showed a significant antitumor effect on KRASmt/aPD1R LLC2 syngeneic-tumors. The antitumor effect was correlated with increased infiltration of human cytotoxic-T, HLA-DR+DC, CD11c+DC, and downregulation of myeloid and regulatory-T cells in TME. Antitumor effect was abolished upon in-vivo depletion of CD8-T, macrophages, and CD4-T cells whereas remained unaffected upon NK-cell depletion. A distinctive protein-expression profile was found after NPRL2 treatment. IFNγ, CD8b, and TBX21 associated with T-cell functions were significantly increased, whereas FOXP3, TGFB1/B2, and IL-10RA were strongly inhibited by NPRL2. A list of T-cell co-inhibitory molecules was also downregulated. Restoration of NPRL2 exhibited significantly slower tumor growth in humanized-mice, which was associated with increased presence of human cytotoxic-T, and DC and decreased percentage of Treg, MDSC, and TAM in TME. NPRL2-stable cells showed a substantial increase in colony-formation inhibition and heightened sensitivity to carboplatin. Stable-expression of NPRL2 resulted in the downregulation of MAPK and AKT-mTOR signaling. Taken-together, NPRL2 gene-therapy induces antitumor activity on KRAS/STK11mt/aPD1R tumors through DC-mediated antigen-presentation and cytotoxic immune-cell activation.
-
- Biochemistry and Chemical Biology
- Cancer Biology
Cancer cells display high levels of oncogene-induced replication stress (RS) and rely on DNA damage checkpoint for viability. This feature is exploited by cancer therapies to either increase RS to unbearable levels or inhibit checkpoint kinases involved in the DNA damage response. Thus far, treatments that combine these two strategies have shown promise but also have severe adverse effects. To identify novel, better-tolerated anticancer combinations, we screened a collection of plant extracts and found two natural compounds from the plant, Psoralea corylifolia, that synergistically inhibit cancer cell proliferation. Bakuchiol inhibited DNA replication and activated the checkpoint kinase CHK1 by targeting DNA polymerases. Isobavachalcone interfered with DNA double-strand break repair by inhibiting the checkpoint kinase CHK2 and DNA end resection. The combination of bakuchiol and isobavachalcone synergistically inhibited cancer cell proliferation in vitro. Importantly, it also prevented tumor development in xenografted NOD/SCID mice. The synergistic effect of inhibiting DNA replication and CHK2 signaling identifies a vulnerability of cancer cells that might be exploited by using clinically approved inhibitors in novel combination therapies.
