SSB REPAIR · Molecular analysis of single-strand break repair (SSBR) in human cells

Molecular analysis of single-strand break repair (SSBR) in human cells

A major deficit of the previous work in this area of SSBR has been the inability to study the repair of and protein recruitment at a site-specific SSB in human chromosomes. Consequently, previous studies have analysed global SSBR events at thousands of heterogeneous SSBs, simultaneously. We decided to attempt the development of systems to induce site-specific SSBs in human cells. Despite successfully validating the constructs in vitro and generating mammalian cell lines harbouring the systems, our attempts to detect SSB induction were completely unsuccessful. We concluded that the levels of induction are below detection levels, and therefore not valid for our intended analysis. We therefore began to investigate the role key new components of SSBR, discovered during the course of the project. APLF was recently identified as a novel component of DNA strand break repair reactions. Indeed, APLF depletion results in single- and double-strand break repair defects. We have conducted through functional analyses of APLF and published this work in a first author publication in Molecular and Cellular Biology in 2008. We concluded that APLF can accumulate at sites of chromosomal damage via a novel zinc finger-dependent mechanisms by binding to poly ADP-ribose; a polymeric molecules synthesised at site of DNA damage. The recent use of inhibitors of PAR synthesis in cancer chemotherapy highlights the socio-economic relevance of advances in our understanding of PAR metabolism. In addition, we undertook a search for new SSBR genes, focusing on the repair of Top1-mediated SSB lesions, a special type of SSB in which the enzyme remains covalently attached to the 3' end of the break (Top1 cleavage complex). This is an important source of endogenous DNA damage which repair is impaired in SCAN1 neurodegenerative disease by a mutation in tyrosyl DNA phosphodiesterase 1 (TDP1). Although TDP1 is the only known tyrosyl DNA phosphodiesterase activity, repair in the absence of functional TDP1 is only impaired but not completely abolished, suggesting the existence of redundant functions for Top1 removal. This agrees with the relatively mild symptoms and late onset of SCAN1 when compared to other hereditary ataxias. A combined strategy of genetics and biochemistry allowed us to identify TTRAP, a member of the Mg2+/Mn2+-dependent family of phosphodiesterases, as novel human tyrosyl DNA phosphodiesterase activity. Although displaying detectable activity on 3' (similar to that of TDP1), TTRAP is fundamentally a 5'-tyrosyl DNA phosphodiesterase, an activity that had not been previously identified in human cells. Consistent with this biochemical function, TTRAP facilitates repair of DSBs induced by topoisomerase II (Top2) in human cells. In the light of these findings, TTRAP is now officially denoted by the Human Genome Nomenclature Organisation (HUGO) as tyrosyl DNA phosphodiesterase-2 (TDP2). TDP1 and TTRAP/TDP2 are therefore complementary enzymatic activities, providing human cells with an ability to cleave both 3'- and 5'- phosphotyrosyl termini at sites of topoisomerase damage. This work was published in Nature, in 2009. The induction of topoisomerase-mediated DNA breaks underlies the clinical efficacy of the so-called topoisomerase poisons, which are widely used in cancer chemotherapy. This work will impact both on our understanding of cancer development and treatment and identifies a possible new target for small molecule intervention during cancer therapy.

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