Investigation and characterisation of novel poly(ADP-ribose)-binding proteins in Dictyostelium discoideum

• Main Author: Gunn, Alasdair
• Other Authors: Lakin, Nicholas
• Published: University of Oxford 2015
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.712521

The genome is under continual assault from endogenous and exogenous sources of DNA damage. The cell has therefore evolved a number of pathways to identify, signal, and ultimately repair DNA lesions. Poly(ADP-ribosyl)ation, the addition of multiple ADP-ribose moieties to proteins to form poly(ADP-ribose) (PAR) chains, has been implicated in several DNA repair pathways. One function of these PAR chains is to act as a scaffold for the recruitment of downstream repair proteins, suggesting the existence of protein domains that specifically bind PAR. Several of these domains have been identified, the best characterised being the PBZ and macro domains. We utilise in silico genome-wide searches to identify novel proteins containing PAR-binding domains in the simple eukaryotic model organism Dictyostelium discoideum. We identity three proteins with unannotated macro domains in Dictyostelium: DNA ligase III (Lig3), an aprataxin-like protein (APL), which also contains a PBZ domain, and Q54R54. The macro domain of APL was found to be circularly permuted compared to the other human and Dictyostelium macro domains; however, structure prediction by homology modelling indicated that it had retained the structure of a classical macro domain. We performed in vitro PAR-binding assays that indicated that the macro domains of both Lig3 and APL bind to PAR chains. Lig3 is enriched on DNA following the infliction of base damage, indicating its role in the SSBR pathway. However, the dependence of this pathway on PARylation has not been determined. In contrast, APL was found to be enriched on chromatin in response to DNA inter-strand cross-links and S-phase-associated double-strand breaks, which was reduced following mutation of both the PBZ and macro domains of APL. Furthermore, APL was found to be mono-ubiquitinated in response to DNA inter-strand cross-links, an event that is dependent on its macro domain region. These data indicate a role for APL in the repair of DNA inter-strand cross-links, or S-phase associated DNA damage, which would not be predicted from its homology to aprataxin, thereby suggesting a protein with novel characteristics in the DDR in Dictyostelium.