Using a well-characterized esiRNA library 
we performed a genome-scale RNAi screen and identified 61 genes that reproducibly decreased or increased the frequency of DNA repair in an assay for homologous recombination 
. Secondary assays for processes relevant to DNA repair corroborated many of the initial findings. Hence, we provide a dataset that should accelerate the discovery of novel genes with roles in DNA repair and associated medical conditions. Eighteen out of the 61 candidate genes have been described in other large-scale mammalian DNA repair studies 
, demonstrating the effectiveness of our screen, but also highlighting that the use of different assays can uncover novel players. Hence, we predict that the development of alternative DNA repair assays for RNAi screens will reveal additional genes implicated in DNA repair. For our screen we co-transfected the “DNA damaging reagent,” I-SceI, together with the esiRNA silencing triggers. Hence, proteins with long half-lives may have been missed in this screen. Assays in which the DSB is introduced some time after the cells were transfected with the silencing triggers could uncover additional genes playing a role during DNA repair.
To prioritize the molecular investigation of the uncharacterized proteins identified in the screen, we employed a structural bioinformatics approach. Based on the prediction that KIAA0415 represents a novel putative helicase we investigated this gene in more detail. Tagging of the gene using the TransgeneOmics approach revealed nuclear as well as cytoplasmic localization and physical interaction with at least four proteins. Investigations of the interaction partners showed that at least two of these proteins are also required for efficient HR-DSBR. Possibly, these proteins form a complex that is required for efficient HR-DSBR. Consequently, the complex would lose its activity when one of the three proteins is depleted.
Two of the interaction partners of KIAA0415 are encoded by genes that are associated with spastic paraplegia. This result prompted us to examine whether KIAA0415 mutations can explain spasticity in patient samples not linked with mutations in any of the known spastic paraplegia genes. We report a homozygous mutation in KIAA0415, responsible for the spastic paraplegia observed in two siblings. Hence, we identify KIAA0415 as a novel spastic paraplegia associated gene. Based on this finding, we propose to rename KIAA0415 to SPG48 according to the HUGO nomenclature. The fact that three proteins that form a protein complex result in similar phenotypic consequences argues that the whole complex is exerting an important function, which is disturbed when one of the proteins is missing or non-functional. It will therefore be interesting to investigate the remaining interaction partners, C20orf29 and DKFZp761E198, for possible mutations in HSP patients, even though they do not map to known HSP loci 
. Although only demonstrated for one case, cell lines derived from a patient carrying a SPG48 mutation were more sensitive to DNA damaging drugs than control cells, corroborating a role of SPG48 in DNA repair. Unfortunately, material from other patients with SPG48 mutations was not available. However, we propose that in the future HSP patients be screened for mutations in SPG48 and that cells from these individuals be checked for DNA repair defects.
Genes mutated in HSP have been associated with several biological functions, including intracellular transport, axonal pathfinding, mitochondrial functions, cholesterol metabolism, myelin formation/stability, and chaperonin activity 
. Based on our findings, we propose that HSP might also be a result of impaired DNA repair, adding HSP to the growing list of neurodegenerative diseases caused by DNA repair deficiencies 
. In agreement with this hypothesis, SPG11 has recently been reported to be phosphorylated upon DNA damage by ATM or ATR 
. Whether SPG48 (and its associated proteins) is a direct component of the HR-DSBR pathway or more indirectly linked to DNA repair remains to be established. Biochemical analysis of the putative helicase domain of SPG48 appears to be an attractive entry point into gaining mechanistic insights into the DNA repair function(s) of SPG48.
The technological advances in RNAi screening have increased the speed at which phenotypic data can be obtained. However, interpretation of the resulting genotype-phenotype relationships remains challenging, and approaches that help to decipher the screening data are highly desirable. Approaches that analyze phenotypic data from unrelated RNAi screens and that combine phenotypic- with localization- and proteomic data 
have been used successfully to bootstrap phenotype-to-function analyses. Here, we explored the possibility of combining RNAi screening data with structural bioinformatics approaches. The obtained results demonstrate that this combination generates valuable information, which helps to prioritize the follow-up studies of uncharacterized candidate genes. We envision that the design of an automatic pipeline to analyze possible structural and functional features beyond protein sequence similarities will further accelerate the characterization of genes identified in RNAi screens. In the future, it will be important to combine the different “omics” and bioinformatics approaches to understand DNA repair at a systems level and to further accelerate the discovery of genes relevant to human pathology.