Excision Repair of UV-Damaged DNA
Excision repair was discovered in the 1960s, and its molecular mechanism was elucidated in the 1980s in E. coli and in the 1990s in humans. Initial studies, first in E. coli and then human cells, revealed that in cells labeled with 3H-thymidine, exposure to UV irradiation followed by an incubation period resulted in transfer of radiolabel from the acid-insoluble high molecular weight DNA fraction to the acid-soluble oligonucleotide fraction of cells.2-4 These findings, together with chromatographic approaches examining the nature of the soluble material, suggested that CPDs were essentially “cut out” (excised) and released from duplex DNA in the form of short, single-stranded (ssDNA) oligonucleotides. UV-sensitive bacterial strains and XP patient cell lines, which were later identified to contain mutations in nucleotide excision repair genes, failed to release radiolabeled thymidine into the acid-soluble fraction.2,3,5 Thus, the ability of both bacterial and human cells to remove CPD-containing oligonucleotides from their genomes seemed to be correlated with their sensitivity to UV radiation as measured by mutation rate and clonogenic survival.
The release of dimer-containing oligonucleotides from duplex DNA is expected to leave a corresponding ssDNA tract (gap) in the duplex that could be restored to a fully double-stranded state through DNA synthesis (patch) across the gap. Coincident with the measurement of dimer release studies, investigations using density labeling and centrifugation and radiolabeling and autoradiography identified the predicted non-conservative mode of DNA replication in UV-treated E. coli and human cells, respectively.6,7 Now termed unscheduled DNA synthesis (UDS), repair replication or repair resynthesis, this replication mechanism (and hence excision repair) was further shown to be defective in human patients with XP.8 In fact, XP genes can be legitimately considered the first tumor suppressor genes to have been identified. These and subsequent studies on UV excision repair led to the following consensus model9: the UV damage is recognized by a repair endonuclease that makes an incision 5′ to the photoproduct; a 5′ to 3′ exonuclease releases the dimer in oligonucleotides 4-6 nt in length; and the resulting gap may or may not be enlarged by exonuclease action before being refilled and ligated to produce the repair patch.
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The identification and cloning of the genes responsible for excision repair and the development of in vitro repair assays with both cell-free extracts and purified proteins ultimately led to reconstitution of both E. coli and human nucleotide excision repair with purified components and elucidation of molecular mechanisms of excision repair in both organisms.10-12 Importantly, these studies revealed that, contrary to the prevailing views at the time, CPDs are removed by concerted dual incisions (rather than endonuclease/exonuclease action) in the form of 12-13 nt-long oligomers in E. coli10 and 24-32 nt-long oligomers (“canonical 30-mer”) in humans.13 Interestingly, though the principle of excision and repair synthesis are nearly identical in these two divergent organisms, the specific proteins that perform the damage recognition and excision steps are not conserved from prokaryotes to eukaryotes. A schematic of the human nucleotide excision repair system and its core repair factors is shown in Figure 2. Comprehensive reviews of nucleotide excision repair have been published,14-16 and the human repair system will be described only briefly here. Recognition of thymine dimers occurs by stochastic order assembly of RPA, XPA and XPC-TFIIH at sites of damage, and specificity is achieved in part by cooperative protein-protein interactions among these factors and mainly by the kinetic proofreading activity of TFIIH. Helicase action by the XPD subunit of TFIIH generates a bubble around the photoproduct and creates the requisite branched DNA substrates for the structure-specific endonucleases XPF and XPG, which incise the damaged strand 20 ± 5 phosphodiester bonds 5′ and 6 ± 3 phosphodiester bonds 3′ of the lesion, respectively. These dual incision events therefore generate an oligonucleotide 24-32-nt in length (also known as the “canonical 30-mer”) that dissociates from the duplex. Release of the canonical 30-mer leaves a canonical 30-mer gap that is then filled in and ligated by a DNA polymerase and ligase. Though the DNA damage recognition, dual incision and repair synthesis steps of nucleotide excision repair have been examined in considerable detail, the fate of the excised canonical 30-mer has not been explored to any significant extent. In our recent study,17 we addressed three interrelated questions: (1) How is the canonical 30-mer released; (2) Does the canonical 30-mer constitute a signal for an intracellular signaling pathway; and (3) What is the ultimate fate of the canonical 30-mer and, most intriguingly, of the cyclobutane pyrimidine dimer? Our study has answered some of these questions and has provided the conceptual framework for answering the others.
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