Defective DNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1

Abstract

Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1). In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells. Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype. Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8). Here we show that in human cells TDP1 is required for repair of chromosomal single-strand breaks arising independently of DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIalpha and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance of genetic integrity in post-mitotic neurons.

TDP2 protects transcription from abortive topoisomerase activity and is required for normal neural function

“…Full-length TTRAP ORF, flanked by NdeI and BamHI restriction sites, was cloned into pCRII-TOPO (Invitrogen) after PCR amplification from pACT-TTRAP-2 using the following primers (Novagen) constructs expressing TDP1 were previously described 6 . pGBKT7 (Clontech) and pET16b (Novagen) constructs expressing wild-type TTRAP, TTRAP E152A , and TTRAP E262A were created by sub-cloning the TTRAP ORF from the appropriate pCRII-TOPO construct using NdeI and EcoRI.…”

“…Gel-purified oligonucleotides were 5'-labelled with 32 P using Alkaline single-cell agarose gel electrophoresis (comet) assays DNA strand breaks were quantified using the alkaline comet assay as described previously 6 .…”

“…2a, inset). This is an established substrate for TDP1 that mimics the Top1-linked SSBs induced by CPT 6,8 . As expected, TDP1 cleaved the 3'-phosphotyrosyl bond and thereby converted the 3'-tyrosine terminus to a 3'-phosphate (Fig.2a, lane 5).…”

“…Surprisingly, however, a complementary human enzyme that cleaves 5'-phosphotyrosyl bonds has not been reported, despite the impact of DNA double-strand breaks harbouring such termini on chromosome instability and cancer [6][7][8] .…”

A novel nonsense mutation in the APTX gene associated with delayed DNA single‐strand break removal fails to enhance sensitivity to different genotoxic agents

“…Two of the proteins involved in the end-processing step are APTX and TDP1 (Caldecott, 2008), involved in neurodegenerative disease, AOA1 (Date et al, 2001;Moreira et al, 2001), and SCAN1 (Takashima et al, 2002), respectively. As previously shown for TDP1 (Takashima et al, 2002), recent studies indicate that aprataxin catalyzes the hydrolytic release of DNA end-blocking lesions with APTX being specific for end-blocking 5'-adenylate and TDP1 for 3'-tyrosyl termini (El-Khamisy et al, 2005;Rass et al, 2007a;Rass et al, 2007b;Rass et al, 2008). A clear defect in cellular SSB repair, however, has only been reported for mutations in TDP1, as the attempts to demonstrate a similar defect in AOA1 failed to give consistent results.…”