CABIMER

 

 

DNA topoisomerases are conserved nuclear enzymes that regulate DNA topology by transiently cleaving and resealing the DNA molecule, fulfilling a fundamental role in virtually every aspect of chromosome metabolism. Nevertheless, erroneous or abortive topoisomerase activity can result in persistent DNA strand breaks with the enzyme covalently attached to 3’ or 5’ DNA ends by a phosphotyrosyl bond, an anomalous structure that can compromise cell survival and/or genome integrity with the consequent implications in tumorigenesis. This peculiarity of topoisomerase catalysis also underlies the anticancer efficacy of topoisomerase poisons, which inhibit the re-ligation step of the reaction inducing the formation of DNA breaks that preferentially target highly proliferating and/or repair defective tumour cells. In addition to this link with cancer therapy, defects in the repair of topoisomerase-induced DNA damage have been linked to progressive neurodegenerative disease.

Fully understanding the mechanisms and regulation governing the repair of topoisomerase-induced damage is therefore extremely important to gain new insights into two processes that are a main concern to human health: (a) cancer, both its onset and its therapy, and (b) neurodegenerative disease.

Topoisomerase-linked breaks usually require processing of the enzyme by the proteasome, leaving a short peptide prior to DNA-damage signalling and repair. The following step involves the removal of the covalently linked topoisomerase derived peptide so repair can proceed. This can occur by tyrosyl-DNA phosphodiesterase (TDP) activity, which cleaves the phosphotyrosyl covalent bond linking the topoisomerase to the DNA terminus. TDP1 acts on 3’-phosphotryosyl bonds typical of topoisomerase I-linked breaks, while TDP2, which has recently been identified in our laboratory, displays preferential activity on 5’-phosphotyrosyl bonds typical of topoisomerase II-linked breaks. Thus, TDP1 and TDP2 are complementary enzymatic activities, providing human cells with an ability to cleave both 3’- and 5’- phosphotyrosyl termini at sites of topoisomerase damage.

Alternatively to this specific repair mechanism, covalently linked protein can be removed from DNA ends by nucleolytic cleavage at an internal phosphodiester bond in the DNA molecule. A wide range of nucleases have been proposed to contribute to this pathway in human cells, including the MRN complex, CtIP, ARTEMIS, FEN1 and ERCC1/XPF.

In our working model TDP function would be important to ensure genome integrity by promoting accurate repair of topoisomerase-induced DNA strand breaks. In contrast, the action of nucleases on DNA ends could lead to mutations and chromosomal reorganizations, which could in turn lead to carcinogenesis. Furthermore, the action of TDPs may be particularly relevant for the survival of long-lived postmitotic cells such as neurons in which nucleolytic pathways might be downregulated. Thus, cancer and neurodegeneration could constitute two sides of the same coin, so defects in repair can manifest as cell death and degeneration in non-proliferating tissue, while resulting in genetic instability in proliferating cells.

We combine the use of animal models with biochemistry and molecular and cellular biology techniques to explore different aspects regarding the repair of topoisomerase-induced breaks. We mainly focus on our recently identified tyrosyl DNA phosphodiesterase 2 (TDP2), studying its mechanism of action and regulation with special attention to the possible implications in cancer and neurodegeneration.

As mentioned above, a combined strategy of genetics and biochemistry has recently allowed us to identify TDP2 as a novel human activity involved in the repair topoisomerase-induced DNA breaks.  We plan to use a similar approach to uncover novel functions involved in the repair of oxidative damage, which represents the main source of spontaneous DNA lesions, and whose accumulation is closely linked to aging and degenerative disease.

The mechanisms by which stem cells deal with DNA damage significantly differ from those of differentiated cells. We will take advantage of the facilities and expertise in CABIMER and particularly in the Stem Cells Department to study these differences. We consider this question of crucial importance if cellular therapy is ever to be successfully applied to the treatment of human disorders associated to the accumulation of DNA damage and defects in DNA repair.