DNA synthesis in ataxia telangiectasia

After the discovery that cultured cells from AT patients are hypersensitive to ionizing radiation the suggestion was made that AT-could be the 1 X-ray-analogue 1 of xeroderma pigmentosum. The latter syndrome (XP) is characterized by hypersensitivity to short-wave UV-radiation, caused by a reduced ability to properly remove UV-induced DNA damage. The evidence for a DNA repair defect in AT cells is not as strong as in the case of XP (see section 2.2.5 of this thesis). Different XP patients vary in their clinical and cellular UV-sensitivity, and this variability roughly correlates with the capacity to repair the UV damage in the DNA. in AT apparent differences in gamma-ray induced repair DNA synthesis contrast with a rather uniform pattern of radiohypersensitivity. The rate of DNA replication is affected by low doses of ionizing or non-ionizing radiation. ln 1977 it was shown that UV-induced inhibition of DNA synthesis is persistent in highly UV-sensitive XP cell strains; whereas less UV-sensitive cells showed recovery from this inhibition. [n the hope to find a consistent biochemical defect in AT cells, it was decided to study the effect ~f ionizing radiation on the rate of DNA synthesis in AT cells

DNA structural elements required for ERCC1-XPF endonuclease activity

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination

DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning o

Transcription-associated breaks in Xeroderma Pigmentosum group D cells from patients with combined features of Xeroderma Pigmentosum and Cockayne Syndrome

Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded

Anti-tumour compounds illudin S and Irofulven induce DNA lesions ignored by global repair and exclusively processed by transcription- and replication-coupled repair pathways.

Illudin S is a natural sesquiterpene drug with strong anti-tumour activity. Inside cells, unstable active metabolites of illudin cause the formation of as yet poorly characterised DNA lesions. In order to identify factors involved in their repair, we have performed a detailed genetic survey of repair-defective mutants for responses to the drug. We show that 90% of illudin's lethal effects in human fibroblasts can be prevented by an active nucleotide excision repair (NER) system. Core NER enzymes XPA, XPF, XPG, and TFIIH are essential for recovery. However, the presence of global NER initiators XPC, HR23A/HR23B and XPE is not required, whereas survival, repair and recovery from transcription inhibition critically depend on CSA, CSB and UVS, the factors specific for transcription-coupled NER. Base excision repair and non-homologous end-joining of DNA breaks do not play a major role in the processing of illudin lesions. However, active RAD18 is required for optimal cell survival, indicating that the lesions also block replication forks, eliciting post-replication-repair-like responses. However, the translesion-polymerase DNA pol eta is not involved. We conclude that illudin-induced lesions are exceptional in that they appear to be ignored by all of the known global repair systems, and can only be repaired when trapped in stalled replication or transcription complexes. We show that the semisynthetic illudin derivative hydroxymethylacylfulvene (HMAF, Irofulven), currently under clinical trial for anti-tumour therapy, acts via the same mechanism

The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA insterstrand cross-link-induced double-strand breaks

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination

Neurological symptoms and natural course of xeroderma pigmentosum

We have prospectively followed 16 Finnish xeroderma pigmentosum (XP) patients for up to 23 years. Seven patients were assigned by complementation analysis to the group XP-A, two patients to the XP-C group and one patient to the XP-G group. Six of the seven XP-A patients had the identical mutation (Arg228Ter) and the seventh patient had a different mutation (G283A). Further patients were assigned to complementation groups on the basis of their consanguinity to an XP patient with a known complementation group. The first sign of the disease in all the cases was severe sunburn with minimal sun exposure in early infancy. However, at the time the diagnosis was made in only two cases. The XP-A patients developed neurological and cognitive dysfunction in childhood. The neurological disease advanced in an orderly fashion through its successive stages, finally affecting the whole nervous system and leading to death before the age of 40 years. Dermatological and ocular damage of the XP-A patients tended to be limited. The two XP-C patients were neurologically and cognitively intact despite mild brain atrophy as seen by neuroimaging. The XP-G patients had sensorineural hearing loss, laryngeal dystonia and peripheral neuropathy. The XP-C patients had severe skin and ocular malignancies that first presented at pre-school age. They also showed immunosuppression in cell-mediated immunity. Neurological disease appears to be associated with the complementation group and the failure of fibroblasts to recover RNA synthesis following UV irradiation, but not necessarily to the severity of the dermatological symptoms, the hypersensitivity of fibroblasts to UVB killing or the susceptibility of keratinocytes to UVB-induced apoptosis

The defect in the AT-like hamster cell mutants is complemented by mouse chromosome 9 but not by any of the human chromosomes

X-ray-sensitive Chinese hamster V79 cells mutants, V-C4, V-E5 and V-G8, show an abnormal response to X-ray-induced DNA damage. Like ataxia telangiectasia (AT) cells, they display increased cell killing, chromosomal instability and a diminished inhibition of DNA synthesis following ionizing radiation. To localize the defective hamster gene (XRCC8) on the human genome, human chromosomes were introduced into the AT-like hamster mutants, by microcell mediated chromosome transfer. Although, none of the human chromosomes corrected the defect in these mutants, the defect was corrected by a single mouse chromosome, derived from the A9 microcell donor cell line. In four independent X-ray-resistant microcell hybrid clones of V-E5, the presence of the mouse chromosome was determined by fluorescent in situ hybridization, using a mouse cot-1 probe. By PCR analysis with primers specific for different mouse chromosomes and Southern blot analysis with the mouse Ldlr probe, the mouse chromosome 9, was identified in all four X-ray-resistant hybrid clones. Segregation of the mouse chromosome 9 from these hamster-mouse microcell hybrids led to the loss of the regained X-ray-resistance, confirming that mouse chromosome 9 is responsible for complementation of the defect in V-E5 cells. The assignment of the mouse homolog of the ATM gene to mouse chromosome 9, and the presence of this mouse chromosome only in the radioresistant hamster cell hybrids suggest that the hamster AT-like mutants are homologous to AT, although they are not complemented by human chromosome 11

Pollitt syndrome patients carry mutation in TTDN1

Complete human genome sequencing was used to identify the causative mutation in a family with Pollitt syndrome (MIM #. 275550), comprising two non-consanguineous parents and their two affected children. The patient's symptoms were reminiscent of the non-photosensitive form of recessively inherited trichothiodystrophy (TTD). A mutation in the TTDN1/. C7orf11 gene, a gene that is known to be involved in non-photosensitive TTD, had been excluded by others by Sanger sequencing. Unexpectedly, we did find a homozygous single-base pair deletion in the coding region of this gene, a mutation that is known to cause non-photosensitive TTD. The deleterious variant causing a frame shift at amino acid 93 (C326delA) followed the right mode of inheritance in the family and was independently validated using conventional DNA sequencing. We expect this novel DNA sequencing technology to help redefine phenotypic and genomic variation in patients with (mono) genetic disorders in an unprecedented manner