Zilio, N.; (2010) Insights into the interplay between sumoylation of chromatin-associated proteins and DNA. Doctoral thesis , UCL (University College London).

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Abstract

The maintenance of correct genome sequence is an essential cellular process in which the small ubiquitin-like modifier SUMO plays an important role, probably because its post-translational conjugation to certain proteins can regulate DNA metabolism. The identities of these proteins remain largely unknown, as do how their sumoylation is controlled or what effects their modification has. I was therefore interested in identifying such factors and studying the upstream signals and downstream consequences of their sumoylation, with a particular focus on how DNA may be involved in these processes. To address this problem, I initially used Xenopus laevis egg extracts to isolate and identify SUMO conjugates from replicating chromatin, as it should be enriched for proteins involved in DNA metabolism. I found that progression through S phase, but not genotoxic stress, altered the abundance of chromatin-associated SUMO conjugates. A proteomic analysis of these species during unperturbed and disrupted S phase identified several proteins with a role in DNA metabolism as putative sumoylation substrates. Some of these modification events were confirmed by western blotting and were also shown to be conserved in the budding yeast Saccharomyces cerevisiae. By further investigating the modification of one of the candidates I discovered, i.e. ORC1, the largest subunit of the six-membered origin recognition complex (ORC), I found that all of the ORC subunits were sumoylated. I, however, also observed that manipulating the general levels of sumoylation in either egg extracts or budding yeast did not affect the recognized functions of ORC in DNA replication, thus indicating that sumoylation does not play a significant role in such a process. I therefore focused on the sumoylation of another candidate I found in my screen, the DNA-break sensor poly(ADP-Ribose) polymerase 1, PARP-1. In vitro, I found that sumoylation of both the full-length protein and its DNA-binding domain alone depended on the presence of intact DNA and was strongly inhibited by nicks in the double helix. In vivo, two main sites of sumoylation in PARP-1 were identified. By mutating them and creating a linear PARP-1-SUMO fusion, to mimic a constitutively sumoylated polymerase, I investigated the functions of PARP-1 sumoylation in human cells. I found that the sumoylation of PARP-1 did not affect the protein’s catalytic activity, localization or binding to intact chromatin or nicked DNA. Instead, sumoylation appeared to accelerate PARP-1’s ubiquitylation and subsequent degradation. These observations exemplify how DNA and sumoylation can interplay with each other to control the properties of chromatin-associated proteins. They also suggest that when PARP-1 is associated to single-stranded DNA breaks, and is therefore engaged in DNA repair, it becomes refractory to sumoylation and subsequent degradation. Thus, sumoylation may help cells to distinguish between two functionally distinct subpopulations of PARP-1: one that is not sumoylated because it is bound to, and in the process of repairing, DNA nicks, and one that is sumoylated because it is bound to intact DNA, which could be involved in other processes in which the polymerase plays a role, such as transcription regulation.

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