A DNA repair gene of Caenorhabditis elegans: a homolog of human XPF

The xeroderma pigmentosum complementation group F (XPF) protein is a structure-specific endonuclease in a complex with ERCC1 and is essential for nucleotide excision repair (NER). We report a single cDNA of Caenorhabditis elegans (C. elegans) encoding highly similar protein to human XPF and other XPF members. We propose to name the corresponding C. elegans gene xpf. Messenger RNA for C. elegans xpf is 5'-tagged with a SL2 splice leader, suggesting an operon-like expression for xpf. Using RNAi, we showed that loss of C. elegans xpf function caused hypersensitivity to ultra-violet (UV) irradiation, as observed in enhanced germ cell apoptosis and increased embryonic lethality. This study suggests that C. elegans xpf is conserved in evolution and plays a role in the repair of UV-damaged DNA in C. elegans.     

Coordination of dual incision and repair synthesis in human nucleotide excision repair

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5′ to the lesion by ERCC1‐XPF and 3′ to the lesion by XPG leads to the removal of a lesion‐containing oligonucleotide of about 30 nucleotides. The resulting single‐stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1‐XPF and XPG, we show that the 5′ incision by ERCC1‐XPF precedes the 3′ incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a ‘cut‐patch‐cut‐patch’ mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.     

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.     

DNA repair capacity: inconsistency between effect of over-expression of five NER genes and the correlation to mRNA levels in primary lymphocytes

We have previously shown that high DNA repair capacity protects psoriasis patients against chemically induced basal cell carcinoma [Dybdahl et al. Mutat. Res. 433 (1999) 15-22]. We have used the same study persons to investigate the correlation between expression of eight genes involved in nucleotide excision repair and DNA repair capacity. mRNA levels of XPA, XPB, XPC, XPD, XPF, XPG, CSB and ERCC1 in primary lymphocytes from 33 individuals were quantified by dot-blots and normalized to beta-actin. ERCC1 and XPD mRNA quantities were highly correlated (r=0.89; P<10(-11)) while XPA, XPB, XPC, XPG, XPFand CSB mRNAs were moderately correlated (r=0.2-0.7). Thus, the mRNA expressions seem to fall in at least two groups. There was a three to sevenfold variation in the expression levels of the mRNAs. This is in contrast to the more than a hundredfold variation in mRNA levels reported in cancer patients.DNA repair capacity was measured in a host cell reactivation assay, where primary lymphocytes were transfected with an UV-irradiated plasmid encoding firefly-luciferase. Only ERCC1 and XPD mRNA levels correlated with the DNA repair capacity (P<0.03). In order to see if ERCC1 or XPD activity was limiting for DNA repair, we cotransfected with plasmids encoding NER genes, thus over-expressing either XPB, XPC, XPD, CSB or ERCC1 in the host cell reactivation assay. Only XPB over-expression increased DNA repair capacity. Thus, there is no indication that neither XPD nor ERCC1 limits the DNA repair capacity. However, our results indicate that ERCC1 and XPD mRNA levels may be used as a proxy for DNA repair capacity in lymphocytes.     

ERCC1/XPF limits L1 retrotransposition

Retrotransposons are currently active in the human and mouse genomes contributing to novel disease mutations and genomic variation via de novo insertions. However, little is known about the interactions of non-long terminal repeat (non-LTR) retrotransposons with the host DNA repair machinery. Based on the model of retrotransposition for the human and mouse LINE-1 element, one likely intermediate is an extension of cDNA that is heterologous to the genomic target, a flap intermediate. To determine whether a human flap endonuclease could recognize and process this potential intermediate, the genetic requirement for the ERCC1/XPF heterodimer during LINE-1 retrotransposition was characterized. Reduction of XPF in human cells increased retrotransposition whereas complementation of ERCC1-deficiency in hamster cells reduced retrotransposition. These results demonstrate for the first time that DNA repair enzymes act to limit non-LTR retrotransposition and may provide insight into the genetic instability phenotypes of ercc1 and xpf individuals.     

Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair and modulation of repair gene expression

Repair of UV-induced DNA lesions in terminally differentiated human hNT neurons was compared to that in their repair-proficient precursor NT2 cells. Global genome repair of (6-4)pyrimidine-pyrimidone photoproducts was significantly slower in hNT neurons than in the precursor cells, and repair of cyclobutane pyrimidine dimers (CPDs) was not detected in the hNT neurons. This deficiency in global genome repair did not appear to be due to denser chromatin structure in hNT neurons. By contrast, CPDs were removed efficiently from both strands of transcribed genes in hNT neurons, with the nontranscribed strand being repaired unexpectedly well. Correlated with these changes in repair during neuronal differentiation were modifications in the expression of several repair genes, in particular an up-regulation of the two structure-specific nucleases XPG and XPF/ERCC1. These results have implications for neuronal dysfunction and aging.     

Thermodynamic Cooperativity and Kinetic Proofreading in DNA Damage Recognition and Repair

In humans UV-induced cyclobutane thymine dimers are excised by the joint action of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF?ERCC1. Yet, in vitro assays show that none of these six factors is capable of detectably discriminating thymine dimer-containing DNA from undamaged DNA. We show how two elementary principles in macromolecular recognition, (1) cooperativity and (2) kinetic proofreading, are utilized to confer specificity to the repair system where none exists at the individual repair factor level and enable human cells to excise thymine dimers with a physiologically relevant specificity and at a biologically acceptable rate.     

UV-B light induces an adaptive response to UV-C exposure via photoreactivation activity in Euglena gracilis.

Phytoplankton such as Euglena are constantly exposed to solar light which is used for photosynthesis. Although the solar ultraviolet (UV) induces DNA damage such as cyclobutane-pyrimidine dimers (CPDs), many kinds of living organisms can repair CPDs by photoreactivation (PR) utilizing the near-UV/blue light component in sunlight. Euglena cells are known to possess such PR activity. In the present paper, the formation of CPDs induced by UV-C exposure and the photoreactivation PR repair of these CPDs by UV-A are demonstrated. To clarify the adaptive responses prior UV-B irradiation on PR activity, cells were cultured in the dark or under UV-B light. When the cells were cultured in the dark for 3 d prior to UV-C exposure, PR activity decreased. When the cells were cultured under UV-B light, however, PR activity increased. These results suggest that exposing the cells to UV-B prior to exposure to UV-C induced an adaptive response towards DNA damage caused by UV-C exposure, and this UV-C induced damage was repaired through PR activity.     

Thermodynamic cooperativity and kinetic proofreading in DNA damage recognition and repair

In humans UV-induced cyclobutane thymine dimers are excised by the joint action of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Yet, in vitro assays show that none of these six factors is capable of detectably discriminating thymine dimer-containing DNA from undamaged DNA. We show how two elementary principles in macromolecular recognition, (1). cooperativity and (2). kinetic proofreading, are utilized to confer specificity to the repair system where none exists at the individual repair factor level and enable human cells to excise thymine dimers with a physiologically relevant specificity and at a biologically acceptable rate.     

Nucleotide excision repair endonuclease genes in Drosophila melanogaster

Nucleotide excision repair (NER) is the primary pathway for the removal of ultraviolet light-induced damage and bulky adducts from DNA in eukaryotes. During NER, the helix is unwound around the damaged site, and incisions are made on the 5' and 3' sides, to release an oligonucleotide carrying the lesion. Repair synthesis can then proceed, using the intact strand as a template. The incisions flanking the lesion are catalyzed by different structure-specific endonucleases. The 5' incision is made by a heterodimer of XPF and ERCC1 (Rad1p-Rad10p in Saccharomyces cerevisiae), and the 3' incision is made by XPG (Rad2p in S. cerevisiae). We previously showed that the Drosophila XPF homologue is encoded by the meiotic recombination gene mei-9. We report here the identification of the genes encoding the XPG and ERCC1 homologues (XPG(Dm) and ERCC1(Dm)). XPG(Dm) is encoded by the mus201 gene; we found frameshift mutations predicted to produce truncated XPG(Dm) proteins in each of two mus201 alleles. These mutations cause defects in nucleotide excision repair and hypersensitivity to alkylating agents and ultraviolet light, but do not cause hypersensitivity to ionizing radiation and do not impair viability or fertility. ERCC1(Dm) interacts strongly in a yeast two-hybrid assay with MEI-9, indicative of the presumed requirement for these polypeptides to dimerize to form the functional endonuclease. The Drosophila Ercc1 gene maps to polytene region 51D1-2. The nucleotide excision repair gene mus210 maps nearby (51E-F) but is distinct from Ercc1.     

Primary mouse fibroblasts deficient for c-Fos, p53 or for both proteins are hypersensitive to UV light and alkylating agent-induced chromosomal breakage and apoptosis

The important regulatory proteins, c-Fos and p53 are induced by exposure of cells to a variety of DNA damaging agents. To investigate their role in cellular defense against genotoxic compounds, we comparatively analysed chromosomal aberrations and apoptosis induced by ultraviolet (UV-C) light and the potent alkylating agent methyl methanesulfonate (MMS) in primary diploid mouse fibroblasts knockout for either c-Fos or p53, or double knockout for both genes. We show that c-Fos and p53 deficient fibroblasts are more sensitive than the corresponding wild-type cells as to the induction of chromosomal aberrations and apoptosis. Double knockout fibroblasts lacking both c-Fos and p53 are viable and were even more sensitive, showing additivity of the chromosomal breakage effects observed in the single knockouts. Regarding the endpoint apoptosis, double knockout fibroblasts displayed a sensitivity similar to c-Fos and p53 deficient cells. The data indicate that (a) both c-Fos and p53 are involved in cellular protection against the clastogenic effect of genotoxic agents, (b) p53 is not required for induction of apoptosis by UV light and MMS, but rather prevents fibroblasts from undergoing apoptotic cell death upon DNA damage, and (c) c-Fos and p53 seem to act independently in determining genotoxic resistance, which is hypothesized to be achieved by impaired DNA repair or differential cell cycle check point control.     

Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position -3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position -6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position -19 to +8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.