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Review
. 2021 Jul:103:103126.
doi: 10.1016/j.dnarep.2021.103126. Epub 2021 Apr 20.

Transcription-coupled nucleotide excision repair: New insights revealed by genomic approaches

Mingrui Duan  1 , Rachel M Speer  2 , Jenna Ulibarri  1 , Ke Jian Liu  2 , Peng Mao  3
Affiliations
Review

Transcription-coupled nucleotide excision repair: New insights revealed by genomic approaches

Mingrui Duan et al. DNA Repair (Amst). 2021 Jul.

Abstract

Elongation of RNA polymerase II (Pol II) is affected by many factors including DNA damage. Bulky damage, such as lesions caused by ultraviolet (UV) radiation, arrests Pol II and inhibits gene transcription, and may lead to genome instability and cell death. Cells activate transcription-coupled nucleotide excision repair (TC-NER) to remove Pol II-impeding damage and allow transcription resumption. TC-NER initiation in humans is mediated by Cockayne syndrome group B (CSB) protein, which binds to the stalled Pol II and promotes assembly of the repair machinery. Given the complex nature of the TC-NER pathway and its unique function at the interface between transcription and repair, new approaches are required to gain in-depth understanding of the mechanism. Advances in genomic approaches provide an important opportunity to investigate how TC-NER is initiated upon damage-induced Pol II stalling and what factors are involved in this process. In this Review, we discuss new mechanisms of TC-NER revealed by genome-wide DNA damage mapping and new TC-NER factors identified by high-throughput screening. As TC-NER conducts strand-specific repair of mutagenic damage, we also discuss how this repair pathway causes mutational strand asymmetry in the cancer genome.

Keywords: CPD-seq; CSB; DNA damage; Mutagenesis; RNA Pol II.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
TC-NER in mammalian cells. RNA Pol II stalling at a DNA lesion leads to the binding of CSB, which recruits CSA and UVSSA. UVSSA then promotes TFIIH recruitment. The elongation factor ELOF1 and the serine/threonine kinase STK19 are also important for TC-NER, but their roles remain elusive. The helicases in TFIIH unwind the two strands of damaged DNA to facilitate dual incision by ERCC1-XPF and XPG on the 5’ and 3’ sides of the lesion, respectively. The excised DNA fragment containing the lesion is bound by TFIIH and released. The gap on the damaged strand is filled by DNA polymerases and DNA ligases. Transcription can be resumed after repair of the damage.
Figure 2.
Figure 2.
Schematic representation of DNA damage/repair mapping methods. (A) XR-seq is a method that maps excision repair products (~30 nt), which are bound by TFIIH in the cell. These short fragments are extracted from cell lysates and ligated to sequencing adaptors. Fragments containing damage are purified by damage immuneprecipitation (IP) using a DNA damage-specific antibody. After damage repair or bypass with a translesion synthesis (TLS) DNA polymerase, the DNA fragment is used for PCR amplification. (B) CPD-seq and NMP-seq are used for mapping UV damage (i.e., CPDs) and alkylation lesions such as 3-meA and 7-meG. Red asterisks represent DNA damage. Damage is cleaved with DNA repair enzymes to generate a new 3’-OH group, which is ligated to a sequencing DNA adaptor (2nd adaptor).
Figure 3.
Figure 3.
Role of Rad26 in yeast TC-NER revealed by CPD-seq analysis. (A) Repair of CPDs in wild-type cells at 2h. Yeast genes (n = 5,205) [74] were aligned at their transcription-start site (TSS) and the fraction of remaining CPDs at 2h (CPD-2h normalized to CPDs-0h) was plotted in DNA regions around the TSS. Transcribed strand (TS) and non-transcribed strand (NTS) are analyzed separately. CPD-seq data was downloaded from Gene Expression Omnibus (accession code GSE145911) [59] and reanalyzed in transcribed regions near the TSS (from −150bp upstream to 450bp downstream of the TSS). The gray background depicts yeast nucleosome occupancy generated with published MNase-seq data [75]. (B) Repair of CPDs in Rad26-deficient cells (i.e., rad26Δ) at 2h in yeast genes. (C) Model depicting the variable requirements for Rad26 in yeast TC-NER in different chromatin regions (e.g., +1 and downstream nucleosomes), and its correlation with the occupancy of transcription elongation factor Spt4-Spt5 and initiation/repair factor TFIIH.
Figure 4.
Figure 4.
Role of TC-NER in causing mutational strand asymmetry. Upper panel: Damage (e.g., UV-induced TC dimer) located on the transcribed strand (TS) stalls Pol II elongation and is repaired by TC-NER. The fast repair leads to low C>T mutation frequency on the TS. Lower panel: A TC dimer located on the non-transcribed strand (NTS) cannot be repaired by TC-NER and is left for repair by GG-NER. The slow repair by GG-NER causes high C>T mutation frequency on the NTS when the damage is replicated by a DNA polymerase.
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