Fission Yeast Rnf4 Homologs Are Required for DNA Repair

Abstract

We describe two RING finger proteins in the fission yeast Schizosaccharomyces pombe, Rfp1 and Rfp2. We show that these proteins function redundantly in DNA repair. Rfp1 was isolated as a Chk1-interacting protein in a two-hybrid screen and has high amino acid sequence similarity to Rfp2. Deletion of either gene does not cause a phenotype, but a double deletion (rfp1⌬rfp2⌬) showed poor viability and defects in cell cycle progression. These cells are also sensitive to DNA-damaging agents, although they maintained normal checkpoint signaling to Chk1. Rfp1 and Rfp2 are most closely related to human Rnf4, and we showed that Rnf4 can substitute functionally for Rfp1 and/or Rfp2. The double mutants also showed significantly increased levels of protein SUMOylation, and we identified an S. pombe Ulp2/Smt4 homolog that, when overexpressed, reduced SUMO levels and suppressed the DNA damage sensitivity of rfp1⌬ rfp2⌬ cells.The fidelity of cell cycle progression is paramount for tissue homeostasis and development and avoidance of disease. A major contributor to this fidelity are the plethora of responses that detect damage to DNA, ensure its repair, and activate checkpoints that modify the cell cycle to allow time for these events to occur. These responses are rapid, highly conserved, and frequently regulated by cascades of post-translational modifications that control the activities of repair proteins and signal transducers of the checkpoints.The fission yeast Schizosaccharomyces pombe is an important model system for the dissection of the molecular controls over DNA damage responses (1, 2). S. pombe has a prominent G 2 period in vegetative cell cycles that represents ϳ70% of total cell cycle time (3). The DNA damage checkpoint functioning during this period signals to activate the Chk1 protein kinase, which in turn prevents entry into mitosis through modulating the Cdc2 regulators Wee1 and Cdc25 (4 -6). Chk1 activation is controlled by reversible phosphorylation on Ser-345 by Rad3 (7-9), an ATR (ataxia telangiectasia-and Rad3-related) homolog, although the mechanism by which this activation occurs is largely obscure. ATR homologs also phosphorylate a number of other proteins in this pathway (1, 10).In addition to phosphorylation cascades, other post-translational modifications regulate DNA damage response pathways.Among these are the covalent modification of proteins by ubiquitin and its related protein, SUMO (small ubiquitin-like modifier). In addition to directing the proteolysis of its targets, ubiquitination can directly influence responses to DNA damage. A well documented example of this is through the post-replication repair proteins that control the mono-and polyubiquitination of proliferating cell nuclear antigen, which in turn enables the bypass of lesions during DNA replication (11-13).SUMO modification of several DNA repair enzymes has also been described (14), although in most cases the molecular effects of such modification remains unclear. Control over protein SUMOylation is achieved by a balance of the activi...

Nuclear organization in genome stability: SUMO connections

“…Similarly, overexpression of the desumoylating enzyme Ulp2 suppresses the DNA damage sensitivity of cells lacking both Rfp1 and Rfp2, the fission yeast functional homologues of Slx5 [54]. Collectively, these results implicate the Slx5/Slx8 STUbL in the maintenance of genome integrity during DNA replication, presumably through the ubiquitylation and subsequent degradation of a SUMO-modified target.…”

TheDrosophilaSTUbL protein Degringolade limits HES functions during embryogenesis

“…S1D,E in the supplementary material) (Abed et al, 2011). RNF4 has also been shown to be a functional ortholog of the Rfp1/Rfp2-Slx8 heterodimer (from now on referred to as Rfp-Slx8) in S. pombe (Kosoy et al, 2007;Prudden et al, 2007) and the Slx5-Slx8 heterodimer in S. cerevisiae (Uzunova et al, 2007). RNF4 and the yeast Rfp-Slx8 and Slx5-Slx8 heterodimers have been shown to be important for DNA repair, kinetochore assembly and genome stability, with the loss of these proteins leading to fragmented chromosomes, elongated nuclei, asymmetric positioning of the nuclei and an accumulation of SUMOylated proteins (Burgess et al, 2007;Kosoy et al, 2007;Prudden et al, 2007;Sun et al, 2007;Mullen and Brill, 2008;Cook et al, 2009;Mukhopadhyay et al, 2010).…”

RNF4 is required for DNA double-strand break repair in vivo

“…18 The yeast and Drosophila orthologues of RNF4 have been implicated in the maintenance of genome stability and DNA repair. 19,20 Because of the early embryonic lethality of Rnf4-null mice, it has not been possible to study the role of Rnf4 in the DNA damage response (DDR) in mammals. Here, we describe a mouse Rnf4 allelic series, which allowed us to establish the first genetic link between Rnf4 and the DDR in mammals.…”