PERSPECTIVES PERSPECTIVES Cell Cycle 11:16, 2997-3002; August 15, 2012; © 2012 Landes Bioscience DNA excision repair Where do all the dimers go? Michael G. Kemp and Aziz Sancar* Department of Biochemistry and Biophysics; University of North Carolina School of Medicine; Chapel Hill, NC USA xposure of cells to UV light from UV causes the formation of pyrimidine Ethe sun causes the formation of dimers, cyclobutane pyrimidine dimers pyrimidine dimers in DNA that have the (CPDs) and pyrimidine-pyrimidone potential to lead to mutation and can- (6–4) photoproducts [(6–4)PPs] between cer. In humans, pyrimidine dimers are adjacent bases in DNA (Fig. 1). These removed from the genome in the form of lesions interfere with both replication and ~30 nt-long oligomers by concerted dual transcription and hence are potentially incisions. Though nearly 50 y of exci- toxic and mutagenic to cells. In humans sion repair research has uncovered many and other placental mammals, the sole © 2012details Landes of UV photoproduct Bioscience. damage mechanism for removing pyrimidine recognition and removal, the fate of the dimers from the genome is nucleotide excised oligonucleotides and, in particu- excision repair. Individuals with the dis- lar, the ultimate fate of the chemically ease xeroderma pigmentosum (XP) have Dovery not stable pyrimidine distribute. dimers remain mutations in genes that encode nucleotide unknown. Physiologically relevant UV excision repair proteins and, as a conse- doses introduce hundreds of thousands quence, have a greater than 5,000-fold of pyrimidine dimers in diploid human increased risk of developing skin cancers.1 cells, which are excised from the genome Furthermore, there are other diseases, within ~24 h. Once removed from the such as systemic lupus erythematosus genome, “where do all the dimers go?” In (SLE), that are characterized by hypersen- a recent study we addressed this question. sitivity to UV and are often precipitated Keywords: UV radiation, DNA repair, Although our study did not determine or exacerbated by exposure to sunlight. nucleotide excision repair, TFIIH, RPA, the fate of the dimer itself, it revealed Therefore, detailed understanding of exci- cell signaling, pyrimidine dimers that the excised ~30-mer is released from sion repair is of importance to human the duplex in a tight complex with the health. Abbreviations: UV, ultraviolet; ssDNA, transcription/repair factor TFIIH. This single-stranded DNA; TFIIH, tran- finding combined with recent reports scription factor II H; RPA, replication Excision Repair that base and oligonucleotide products of protein A; CPD, cyclobutane pyrimidine of UV-Damaged DNA the base and double-strand break repair dimer; ATM, ataxia telangiectasia mutat- pathways also make stable complexes Excision repair was discovered in the ed; MRN, Mre11-Rad50-Nbs1; ATR, with the cognate repair enzymes, and 1960s, and its molecular mechanism was Ataxia telangiectasia-mutated and Rad3- that these complexes activate the MAP elucidated in the 1980s in E. coli and in related; XP, xeroderma pigmentosum; kinase and checkpoint signaling path- the 1990s in humans. Initial studies, first SLE, systemic lupus erythematosus ways, respectively, raises the possibility in E. coli and then human cells, revealed Submitted: 06/07/12 that TFIIH-30-mer excision complexes that in cells labeled with 3H-thymidine, Revised: 06/13/12 may play a role in signaling reactions in exposure to UV irradiation followed by response to UV damage. an incubation period resulted in trans- Accepted: 06/13/12 fer of radiolabel from the acid-insoluble http://dx.doi.org/10.4161/cc.21126 Ultraviolet (UV) radiation from the sun is high molecular weight DNA fraction to *Correspondence to: Aziz Sancar; the most common environmental carcino- the acid-soluble oligonucleotide fraction Email: [email protected] gen that leads to skin cancer in humans. of cells.2-4 These findings, together with www.landesbioscience.com Cell Cycle 2997 Figure 1. DNA base damage induced by UV. Exposure of adjacent thymidine bases in DNA results in formation of (6–4) photoproducts and cyclobu- tane thymidine dimers. chromatographic approaches examining releases the dimer in oligonucleotides branched DNA substrates for the struc- the nature of the soluble material, sug- 4–6 nt in length; and the resulting gap ture-specific endonucleases XPF and XPG, gested that CPDs were essentially “cut may or may not be enlarged by exonucle- which incise the damaged strand 20 ± 5 out” (excised) and released from duplex ase action before being refilled and ligated phosphodiester bonds 5' and 6 ± 3 phos- DNA in the form of short, single-stranded to produce the repair patch. phodiester bonds 3' of the lesion, respec- (ssDNA) oligonucleotides. UV-sensitive The identification and cloning of the tively. These dual incision events therefore bacterial strains and XP patient cell lines, genes responsible for excision repair and generate an oligonucleotide 24–32-nt which were later identified to contain the development of in vitro repair assays in length (also known as the “canonical mutations in nucleotide excision repair with both cell-free extracts and purified 30-mer”) that dissociates from the duplex. genes, failed to release radiolabeled thy- proteins ultimately led to reconstitution Release of the canonical 30-mer leaves a midine into the acid-soluble fraction.2,3,5 of both E. coli and human nucleotide canonical 30-mer gap that is then filled Thus, the ability of ©both bacterial2012 and excision Landes repair with purified Bioscience. components in and ligated by a DNA polymerase and human cells to remove CPD-containing and elucidation of molecular mechanisms ligase. Though the DNA damage recog- oligonucleotides from their genomes of excision repair in both organisms.10-12 nition, dual incision and repair synthesis seemed to be correlated with their sensitiv- Importantly, these studies revealed that, steps of nucleotide excision repair have ity to UV radiation as measured by mutaDo- contrary not to the distribute. prevailing views at the been examined in considerable detail, tion rate and clonogenic survival. time, CPDs are removed by concerted dual the fate of the excised canonical 30-mer The release of dimer-containing oli- incisions (rather than endonuclease/exo- has not been explored to any significant gonucleotides from duplex DNA is nuclease action) in the form of 12–13 nt- extent. In our recent study,17 we addressed expected to leave a corresponding ssDNA long oligomers in E. coli10 and 24–32 three interrelated questions: (1) How is tract (gap) in the duplex that could be nt-long oligomers (“canonical 30-mer”) the canonical 30-mer released; (2) Does restored to a fully double-stranded state in humans.13 Interestingly, though the the canonical 30-mer constitute a signal through DNA synthesis (patch) across the principle of excision and repair synthesis for an intracellular signaling pathway; and gap. Coincident with the measurement are nearly identical in these two divergent (3) What is the ultimate fate of the canon- of dimer release studies, investigations organisms, the specific proteins that per- ical 30-mer and, most intriguingly, of the using density labeling and centrifugation form the damage recognition and excision cyclobutane pyrimidine dimer? Our study and radiolabeling and autoradiography steps are not conserved from prokaryotes has answered some of these questions and identified the predicted non-conservative to eukaryotes. A schematic of the human has provided the conceptual framework mode of DNA replication in UV-treated nucleotide excision repair system and its for answering the others. E. coli and human cells, respectively.6,7 core repair factors is shown in Figure 2. Now termed unscheduled DNA synthesis Comprehensive reviews of nucleotide exci- New Steps in Nucleotide (UDS), repair replication or repair resyn- sion repair have been published,14-16 and Excision Repair thesis, this replication mechanism (and the human repair system will be described hence excision repair) was further shown only briefly here. Recognition of thymine In our study, which in part was motivated to be defective in human patients with dimers occurs by stochastic order assem- by the question “where do all the dimers X P. 8 In fact, XP genes can be legitimately bly of RPA, XPA and XPC-TFIIH at sites go?,” we first wished to know, rather, considered the first tumor suppressor of damage, and specificity is achieved in “where do all the canonical 30-mers go?” genes to have been identified. These and part by cooperative protein-protein inter- To this end, we used an in vitro excision subsequent studies on UV excision repair actions among these factors and mainly assay with a radiolabeled dimer-containing led to the following consensus model9: by the kinetic proofreading activity of model DNA substrate and cell-free extract the UV damage is recognized by a repair TFIIH. Helicase action by the XPD sub- or purified repair factors and identified endonuclease that makes an incision 5' to unit of TFIIH generates a bubble around new steps in excision repair that occur fol- the photoproduct; a 5' to 3' exonuclease the photoproduct and creates the requisite lowing the dual incision event. As shown 2998 Cell Cycle Volume 11 Issue 16 Figure 2. Model of human nucleotide excision repair. UV-induced thymine dimers (T<>T) are recognized by the actions of XPA, RPA, and XPC-TFIIH and DNA is unwound around the dimer by the helicase activity of TFIIH. Following dual incisions by the XPF and XPG nucleases, an oligonucleotide ~30 nt in length is released from the duplex in complex with TFIIH. The remaining gap is filled in by the actions of DNA polymerase (Pol) and DNA ligase (Lig). Release of the excised oligonucleotide from TFIIH recycles TFIIH for new rounds of repair. The released 30-mer can then be targeted for degradation by nucleases or bound by RPA, which limits its degradation. © 2012 Landes Bioscience. in Figure 2, contrary to current models for understanding the efficiency of UV TFIIH and RPA away from gene promot- of excision repair, we discovered that the photoproduct removal from the human ers, replication forks and recombination excised canonical “30-mer” is released genome.