Kunkel on DNA Replication Fidelity

Background: Caenorhabditis elegans is an important model for the study of DNA damage and repair related processes such as aging, neurodegeneration, and carcinogenesis. However, DNA repair is poorly characterized in this organism. We adapted a quantitative polymerase chain reaction assay to characterize repair of DNA damage induced by ultraviolet type C (UVC) radiation in C. elegans, and then tested whether DNA repair rates were affected by age in adults. Results: UVC radiation induced lesions in young adult C. elegans, with a slope of 0.4 to 0.5 lesions per 10 kilobases of DNA per 100 J/m2, in both nuclear and mitochondrial targets. L1 and dauer larvae were more than fivefold more sensitive to lesion formation than were young adults. Nuclear repair kinetics in a well expressed nuclear gene were biphasic in nongravid adult nematodes: a faster, first order (half-life about 16 hours) phase lasting approximately 24 hours and resulting in removal of about 60% of the photoproducts was followed by a much slower phase. Repair in ten nuclear DNA regions was 15% and 50% higher in more actively transcribed regions in young and aging adults, respectively. Finally, repair was reduced by 30% to 50% in each of the ten nuclear regions in aging adults. However, this decrease in repair could not be explained by a reduction in expression of nucleotide excision repair genes, and we present a plausible mechanism, based on gene expression data, to account for this decrease. Conclusion: Repair of UVC-induced DNA damage in C. elegans is similar kinetically and genetically to repair in humans. Furthermore, this important repair process slows significantly in aging C. elegans, the first whole organism in which this question has been addressed. Background In vitro assays, cell culture systems, and simple unicellular organisms continue to be crucial in elucidating mechanistic aspects of the formation and repair of DNA damage. However, the ability to study DNA damage, and especially its repair in vivo, is somewhat limited in metazoans. Studies in mouse models have been very informative, but they are also expensive and time consuming. Caenorhabditis elegans is a Published: 1 May 2007 Genome Biology 2007, 8:R70 (doi:10.1186/gb-2007-8-5-r70) Received: 11 July 2006 Revised: 3 November 2006 Accepted: 1 May 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/5/R70 Genome Biology 2007, 8:R70 R70.2 Genome Biology 2007, Volume 8, Issue 5, Article R70 Meyer et al. http://genomebiology.com/2007/8/5/R70 powerful system that is increasingly used to study many human conditions that are affected by DNA damage and repair, including carcinogenesis [1,2], neurodegenerative diseases [3,4], and aging [5,6]. Homologs of many human DNA genes are present in the C. elegans genome [7,8], suggesting that this simple multicellular eukaryote might be a good model for the study of DNA repair processes in higher eukaryotes. Furthermore, evidence is building that many of these genes are homologous in function as well as sequence; mutations or RNA interference (RNAi) knockdown of apparent DNA repair homologs have produced genotoxin-sensitive phenotypes [7,9-14], and RNAi screens for genes that protect against mutations have identified DNA repair gene homologs in C. elegans [15]. Finally, the molecular pathways that mediate cellular response to DNA damage, including apoptosis, are fairly well conserved between C. elegans and humans [16,17]. Although there are some studies of DNA repair in C. elegans (for review [18,19]), a simple, versatile assay that permits the study of gene-specific damage and repair in this organism has not been described. We adapted a quantitative polymerase chain reaction (QPCR)-based assay [20,21] to detect damage and repair of damage in the nuclear and mitochondrial genomes of C. elegans. Using this assay, we asked two questions: is the repair of DNA damage induced by ultraviolet type C (UVC; 254 nm) in C. elegans comparable to that observed in mammals; and are DNA repair rates different in young and aging populations of C. elegans? In mammals, repair of UVC-induced DNA damage occurs through nucleotide excision repair (NER) [22,23]. NER is operative only in the nucleus, and it is responsible for the removal of a large number of structurally diverse bulky DNA lesions. NER consists of two distinct molecular pathways: global genomic repair (GGR), in which lesions present in any portion of the genome are detected and removed; and transcription-coupled repair (TCR), in which lesions are detected and subsequently removed when they block the progression of RNA polymerase II. If C. elegans homologs of mammalian NER genes function in a similar manner, then loss-of-function mutations in key NER genes would inhibit repair. Furthermore, the repair of highly transcribed regions of the nuclear genome should be faster than that of poorly or nontranscribed regions of the nuclear genome. We tested these predictions, and additionally characterized the kinetics of repair of a well-transcribed nuclear region in order to ask whether the repair kinetics are similar to those observed in mammalian cells in culture. We also asked whether repair of UVC damage is less efficient in the nuclei of aging than in those of young adult C. elegans. There is evidence that nuclear genome integrity may be related to the aging process in mammals [24,25] and that repair rates decline in mammalian cells in culture [25,26]. However, very few in vivo, whole organism data have been reported that address this hypothesis [27]. Furthermore, there is little evidence to support the hypothesis that DNA repair capacity is related to age in C. elegans, despite the extensive use of this organism as a model for aging [5,6]. In this study, we observed a 30% to 50% decrease in DNA repair in aging C. elegans (assayed at 6 days after L4 molt, corresponding to 60% of the population's mean adult lifespan), and then performed gene expression profiling in young and aging adults to generate hypotheses to explain the mechanism of that decline. Results Exposure to UVC radiation causes similar, dosedependent damage in the nuclear and mitochondrial genomes We adapted a QPCR assay for analyzing gene-specific DNA damage and repair to C. elegans. The QPCR assay quantifies DNA damage by utilizing the ability of many DNA lesions to block or inhibit the progression of DNA polymerases [20]. Under quantitative conditions, PCR amplification of large (about 10 to 15 kilobases [kb]) regions of genomic DNA is reduced in damaged samples as compared with less damaged samples. This reduction in amplification can be converted to a lesion frequency by application of the Poisson distribution [28]. The use of PCR methodology permits the detection of nuclear and mitochondrial lesions in nanogram quantities of total genomic DNA. Young adult (24 hours after L4 stage, hereafter referred to as '1-day-old') N2 (wild-type) nematodes exposed to 50, 100, 200, or 400 J/m2 UVC (254 nm) irradiation exhibited a dosedependent increase in lesions, as detected by QPCR (Figure 1). Lesions were induced with a slope of 0.4 to 0.5 lesions/10 kb per 100 J/m2 UVC, with some loss of linearity evident at the higher doses. No difference was observed in lesion induction between nuclear and mitochondrial genomes. The nuclear target used was the DNA polymerase epsilon gene region; the mitochondrial target comprises the majority of the mitochondrial genome (see Materials and methods, below). Additionally, purified human and nematode genomic DNA were exposed to 5, 10, and 20 J/m2 UVC, and damage quantified by QPCR using either previously described human primers (DNA polymerase beta [21]) or nematode DNA polymerase epsilon primers. The dose-response relation was indistinguishable for purified human and nematode genomic DNA (data not shown). Different life stages of C. elegans vary in susceptibility to UVC-induced nuclear and mitochondrial DNA damage Different life stages of N2 or glp-1 nematodes exposed to 0, 100, or 200 J/m2 UVC exhibited marked differences in susceptibility to induction of DNA damage (Figure 2), with starved L1 larvae the most and 1-day-old N2 adults the least susceptible. The glp-1 mutant is deficient in germline proliferation at 25°C [29], and only germline cells undergo division Genome Biology 2007, 8:R70 http://genomebiology.com/2007/8/5/R70 Genome Biology 2007, Volume 8, Issue 5, Article R70 Meyer et al. R70.3