Ubiquitin, SUMO and PAR : deciphering recruitment signals at DNA damage sites

Abstract

Protecting genome integrity is a vital task that is constantly challenged by various genotoxic stresses. Among different types of DNA damage, double-strand breaks (DSBs) constitute a particular threat since they result in the loss of integrity of both complementary DNA strands. To cope with DNA lesions, cells have evolved efficient mechanisms to sense and repair damaged DNA, generally referred to as the DNA damage response (DDR). The DDR is particularly dependent on a multitude of post-translational modifications (PTMs) to facilitate the correct spatial and temporal recruitment of signaling and repair proteins. Among prominent PTMs that have been shown to be involved in the DDR are ubiquitylation, modification with small ubiquitin-like modifier (SUMOylation) and poly(ADP-ribos)(PAR)ylation, which are the topic of this thesis.

Paper I shows that the deubiquitylating enzyme ataxin-3 is recruited to DSBs in a SUMOylation-dependent manner. Ataxin-3 interacts with SUMO1 and its catalytic activity was stimulated in vitro by SUMO1. MDC1, a mediator of the DSB response, was identified as a substrate on which ataxin-3 is counteracting the RNF4 E3 ubiquitin ligase. By preventing ubiquitin-dependent removal, ataxin-3 is prolonging the chromatin retention time of MDC1, which, we propose, may ensure that the response cascade is accurately activated. Indeed, DNA damage-induced ubiquitylation downstream of MDC1 is impaired in the absence of ataxin-3 leading to inefficient recruitment of 53BP1 and BRCA1 and DSB repair. In addition to SUMOylation, the early and transient recruitment of ataxin-3 to DSBs requires DNA damage-induced PARylation, which is presented in paper II. While we did not observe binding of ataxin-3 to PAR chains, ataxin-3 was found to be an interactor and substrate of PARP1. The recruitment of RNF4 to DNA damage was independent of PAR conjugation. The exact recruitment mechanism of ataxin-3 to DSBs by PARylation has not been elucidated at this stage, but the dual recruitment mode requiring SUMO and PAR likely allows spatiotemporal regulation and separates the retention of ataxin-3 from the opposed enzyme RNF4. The recruitment of the cohesin loader NIPBL to sites of DNA damage was investigated in paper III. Two independent recruitment mechanisms of NIPBL were identified that depend on the type of DNA damage. The N-terminus of NIPBL is recruited by interacting with HP1, while ATM/ATR activity mediates the retention of the C-terminus. Both pathways require additional DNA damage-induced ubiquitin signaling. While NIPBL accrual at nuclease-induced DSBs is strictly dependent on its HP1 binding motif, both recruitment mechanisms can compensate each other at laser-inflicted DNA damage.

In summary, work in this thesis presents two proteins, ataxin-3 and NIPBL, which are recruited to sites of DNA damage by combinatorial PTMs. Dual recruitment mechanisms likely enable the retention of DDR components with high spatiotemporal resolution.