Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - TOPOmics (Global dynamics of topoisomerase-induced DNA breaks)

Teaser

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...

Summary

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 tumourigenesis. 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 neurological disease. Understanding the cellular response to topoisomerase-induced breaks is therefore key for important aspects of human health, with possible implications in the development of novel diagnostic, prognostic and therapeutic tools.

This project aims at acquiring a comprehensive picture of the dynamics of topoisomerase-induced DNA breaks: from their occurrence and repair to the consequences for genome expression and integrity. In this period, we have developed novel assays to detect and isolate the different intermediates of topoisomerase-induced break appearance and repair, overcoming major traditional limitations in the field. We are currently using these tools to determine a time-resolved genome-wide distribution of these intermediates, to obtain a global view of the cellular dynamics of topoisomerase-induced DNA breaks. Furthermore, we have performed proteomic and genetic screenings that have allowed us to identify novel factors and pathways specifically involved the cellular response to topoisomerase-induced breaks. We have validated and characterized the molecular role of the most interesting factors identified.

Work performed

In this period, we have obtained the following major results:

- Development of novel methods to identify and isolate different intermediates in the catalytic metabolism of DNA topoisomerases and DNA breaks induced by their aberrant action.
- Genome-wide maps of regions where the different intermediates of topoisomerase function accumulate.
- ZATT (ZNF451) is a novel component of the cellular response to topoisomerase-induced DNA breaks.
- Nucleolytic activities and PIKK kinases of the DNA-damage response coordinate alternative repair of topoisomerase-induced DNA breaks.

Final results

In the following period, we will finish the analysis regarding the genome-wide distribution of topoisomerase function and breaks, integrating the information obtained. We will also extend this analysis to more physiological models, such as specific tissues or cancer models. Finally, we will also continue the characterization of the most interesting identified factors with an emphasis of possible physiological implications.