Molecular mechanisms and genomic maps of DNA excision repair in Escherichia coli and humans

Abstract

Nucleotide excision repair is a major DNA repair mechanism in all cellular organisms. In this repair system, the DNA damage is removed by concerted dual incisions bracketing the damage and at a precise distance from the damage. Here, we review the basic mechanisms of excision repair in Escherichia coli and humans and the recent genome-wide mapping of DNA damage and repair in these organisms at single-nucleotide resolution.

Keywords: carcinogenesis; chemotherapy; damage mapping; damage recognition; kinetic proofreading; repair mapping.

Figure 1.

Molecular mechanisms of nucleotide excision repair. A, E. coli excision repair. In addition to the three core dual incision proteins, photolyase (Phr) aids in recognition of CPDs both in vivo and in vitro and accelerates the rate of CPD repair. Mfd translocase couples transcription to repair, and UvrD helicase releases the excised oligomer, freeing UvrB and UvrC for catalytic turnover (58). B, E. coli excision repair factors. C, human excision repair. In addition to the six core excision repair factors, the DDB heterodimer stimulates repair of CPDs in vivo but not in vitro by a poorly defined mechanism. In TCR, stalled RNAPII with the aid of CSA and CSB acts as the damage sensor and accelerates the rate of repair of the transcribed strand. D, human excision repair factors. * indicates TFIIH subunits not essential for excision repair. B and D, green, core repair factors; purple, transcription-repair coupling factors; blue, other repair proteins involved in excision repair.

Figure 2.

Damage-seq and XR-seq methods for high-resolution genome-wide mapping of DNA damage and repair. A, damage-seq. The key step is the arrest of a high-fidelity DNA polymerase at the nucleotide 3′ to the damaged base (12). B, XR-seq. The key step is the capture of the excised oligomer by TFIIH co-immunoprecipitation followed by damage-specific immunoprecipitation (IP) (11).

Figure 3.

XR-seq analysis of E. coli genome. A, effect of genetic background on recovery of the excised oligomer. In WT cells, 12–13-mers are captured at low yield. In triple exonuclease mutant deficient in major ssDNA exonucleases, the excised oligomer is degraded to a 10-mer by a 3′- to 5′-exonuclease that stops at a nucleotide 3′ to the dimer. Most strikingly, in the uvrD⁻ mutant the excised oligomer is much more abundant than in other strains and almost exclusively 12–13-nt in length, consistent with the idea that the “excised” oligomer is not released from the UvrB-UvrC-DNA complex in the absence of UvrD and hence is protected from ssDNA nucleases. B, frequency distribution of log2-transformed TS/NTS repair in all annotated genes. E. coli genes in three indicated strains are colored by sense strand transcription levels going from the lowest quartile RNA-seq count colored in red, to orange, to green, and to the highest transcription quartile in blue. The log2 (TS/NTS) means for each strain are 1.16 for WT, 0.68 for mfd⁻, and 1.17 for uvrD⁻ mutant. The vertical black line represents the border where TS repair level is equal to NTS repair. C, screen shots of rRNA operon (including gltT RNA gene) transcription (RNA-seq) and excision repair maps (XR-seq) for E. coli WT, and mfd⁻, and uvrD⁻ mutants. Note the reversal of the TS/NTS repair ratio in mfd⁻ versus WT and the enhancement of the TS/NTS ratio in uvrD⁻ mutant even though overall repair is reduced in this strain (58).

Figure 4.

Damage formation and repair maps of the human genome. A, comparison of UV damage distribution in cellular DNA and naked DNA. Screen shots of the indicated coordinates of chromosome 3 are shown (73). B, screen shots of the XR-seq data for the same chromosome 3 coordinates shown in A. Note the transcribed strand-specific repair for CPD both in WT and XP-C mutant cells and in XP-C mutant cells only for (6-4)PP and the absolute dependence of repair of both photoproducts on transcription and only in the transcribed strand. In CS-B mutant there is no effect of transcription on repair of either lesion (11), and transcription does not inhibit repair of template strand lesions as is seen in mfd⁻ E. coli (Fig. 3C). C, chromatin states affect repair rates of cisplatin damage (upper panel) but not cisplatin damage formation (lower panel). Open chromatin states and transcriptionally active regions are repaired more efficiently compared with weakly transcribed regions and heterochromatin (12). D, effect of nucleosomes. Repair efficiency is anti-phase with the nucleosome center in agreement with the in vitro data showing inhibition of cisplatin damage in the nucleosome core (12). E, “volcano pattern” of excision repair of CPDs around active transcription factor-binding sites (TFBS, red bars on x axis), which overlap with DNase I-hypersensitive sites (DHS). Repair is strongly inhibited at the center of the transcription factor-binding sites and is flanked by two peaks of repair in the DNase I-hypersensitive sites, whereas mutation rate “erupts” at the center of the repair “crater” where repair is inhibited (93).