A Genome-Scale DNA Repair RNAi Screen Identifies SPG48 as a Novel Gene Associated with Hereditary Spastic Paraplegia

Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany.
PLoS Biology (Impact Factor: 9.34). 06/2010; 8(6):e1000408. DOI: 10.1371/journal.pbio.1000408
Source: PubMed


DNA repair is essential to maintain genome integrity, and genes with roles in DNA repair are frequently mutated in a variety of human diseases. Repair via homologous recombination typically restores the original DNA sequence without introducing mutations, and a number of genes that are required for homologous recombination DNA double-strand break repair (HR-DSBR) have been identified. However, a systematic analysis of this important DNA repair pathway in mammalian cells has not been reported. Here, we describe a genome-scale endoribonuclease-prepared short interfering RNA (esiRNA) screen for genes involved in DNA double strand break repair. We report 61 genes that influenced the frequency of HR-DSBR and characterize in detail one of the genes that decreased the frequency of HR-DSBR. We show that the gene KIAA0415 encodes a putative helicase that interacts with SPG11 and SPG15, two proteins mutated in hereditary spastic paraplegia (HSP). We identify mutations in HSP patients, discovering KIAA0415/SPG48 as a novel HSP-associated gene, and show that a KIAA0415/SPG48 mutant cell line is more sensitive to DNA damaging drugs. We present the first genome-scale survey of HR-DSBR in mammalian cells providing a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. The discovery that proteins forming a novel protein complex are required for efficient HR-DSBR and are mutated in patients suffering from HSP suggests a link between HSP and DNA repair.

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    • "However, its functional importance was highlighted in a recent study of AP-5 mutations in patients. Notably, mutation in ζ subunit also causes HSP disease [77], indicating AP-4 and AP-5 may sort the same cargo proteins or both important for axonal maintenance [78]. "
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    • "Disruption of spg11 (SPG11, spatacsin) or zfyve26 (SPG15, spastizin) expression during zebrafish development induced a range of developmental defects, including locomotor impairment and abnormal architecture of spinal MN axons (Martin et al., 2012; Southgate et al., 2010). These two proteins are components of a multi-protein complex (Słabicki et al., 2010) and AMOs targeting the two genes at the same time suggested that they were involved in a pathway required for spinal MN axon outgrowth (Martin et al., 2012). However, an EMS (Raldú a et al., 2008) was apparently induced by changing the expression of these genes, thus making it difficult to interpret the phenotype, which may also be due to a developmental delay. "
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    • "High-throughput screening approaches have been mostly confined to searching for genes, which modulate HDR efficiency (17,18,19,20). Such screens are generally based on siRNA techniques and often result in a relatively high false positive rate due to non-specific silencing of genes, as was recently demonstrated (18). "
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