Novel regulators of post-translational modifications in the dna damage response

Resumen

Posttranslational modifications, such as ubiquitination, are important events in the DNA damage response, regulating the localization and degradation of key proteins in the DNA damage response. Although several E3 ubiquitin ligases have been identified to mediate ubiquitination of H2AX and PCNA, less is known about the ubiquitin hydrolases (DUBs) responsible for removing ubiquitin in these two key proteins of the DNA damage response. Herein, we have developed a screening for these DUB enzymes overexpressing plasmids with most human DUBs present in a library and identified the possible candidates. In this screening, DUB3 appeared as a candidate for both, H2AX and PCNA, while SENP5 was also related with regulation of PCNA ubiquitination. Subsequent experiments confirmed that overexpression of wild type DUB3 and SENP5, but not a catalytically inactive version of them, altered the amount of monoubiquitinated H2AX (DUB3) and monoubiquitinated PCNA (DUB3 and SENP5). The following objectives were to verify that these DUBs are indeed necessary for the deubiquitination of H2AX and PCNA. We have studied the functional consequences of H2AX and PCNA deubiquitination through increasing and decreasing the expression levels of DUBs candidates. Finally, we have identified DUB3 and SENP5 as new actors in the DNA damage response by regulating deubiquitination of H2AX and PCNA.