Werner Helicase-interacting Protein 1 Binds Polyubiquitin via Its Zinc Finger Domain

Abstract

DNA repair is regulated on many levels by ubiquitination. In order to identify novel connections between DNA repair pathways and ubiquitin signaling, we used mass spectrometry to identify proteins that interact with lysine 6-linked polyubiquitin chains. From this proteomic screen, we identified the DNA repair protein WRNIP1 (Werner helicase-interacting protein 1), along with nucleosome assembly protein 1, as novel ubiquitin-interacting proteins. We found that a small zinc finger domain at the N terminus of WRNIP1 is sufficient and necessary for noncovalent ubiquitin binding. This ubiquitin-binding zinc finger (UBZ) domain binds polyubiquitin but not monoubiquitin and appears to show no specificity for polyubiquitin chain linkage. A homologous zinc finger domain in RAD18 also binds polyubiquitin, suggesting a wider role for the UBZ domain in DNA repair. The WRNIP1 ubiquitin-binding function, along with its previously established ATPase activity, suggests that WRNIP1 plays a role in the metabolism of ubiquitinated proteins. Supporting this model, deletion of MGS1, the yeast homolog of WRNIP1, slows the rate of ubiquitin turnover, rendering yeast resistant to cycloheximide. We also find that WRNIP1 is heavily modified with ubiquitin and SUMO, revealing complex layers in the involvement of ubiquitin pathway proteins in the regulation of DNA repair. The novel ubiquitin-binding ability of WRNIP1 sheds light on the role of UBZ domain-containing proteins in postreplication DNA repair.

Human Wrnip1 Is Localized in Replication Factories in a Ubiquitin-binding Zinc Finger-dependent Manner

“…It has been recently shown that Wrnip1 can bind to poly-but not mono-ubiquitin (12). To validate these findings, we fused the amino acids 9 -48 spanning the UBZ domain of human Wrnip1 in-frame with GST, expressed such fusion in E. coli, and used it to pull down a mixture of Ub chains varying in length from 2 to 7 monomers, linked either via lysine 48 or 63.…”

“…We focused our attention on Wrnip1/Mgs1, which was recently shown to selectively bind poly-Ub chains and regulate the turnover of ubiquitylated proteins (12). Wrnip1 has been genetically linked to the ubiquitin ligase Rad18 in yeast and chicken cells and proposed to function in response to stalling of DNA replication forks (8,18,19).…”

“…Additionally, purified human Wrnip1 can interact with DNA polymerase ␦ and increase the initiation efficiency of DNA replication in vitro (11). Besides a possible function in DNA replication, human Wrnip1 has also been shown to selectively bind ubiquitin (Ub) chains but not mono-Ub and has been proposed to regulate the turnover of ubiquitylated proteins (12). However, the precise function(s) of Wrnip1 in mammalian cells still remain unclear.…”

Cooperation of replication Protein A with the ubiquitin ligase Rad18 in DNA damage bypass

“…The protein contains a RING domain, essential for its ubiquitin ligase activity, 22 a ubiquitin-binding zinc finger (UBZ) motif 23,24 and a SAP domain, which has been shown to mediate DNA binding in many other proteins. 25,26 Recently, the DNA-binding properties of hRad18 have been characterized in detail.…”

The discovery of a Werner helicase interacting protein (WHIP) association with the nuclear pore complex

“…33 WHIP may be a modulator for initiation or re-initiation events during DNA polymerase delta-mediated DNA synthesis and with an intrinsic ATPase activity that may function as a sensor of DNA damage or of arrested replication forks and regulates the extent of DNA synthesis. 36,43,44 Mutations of the yeast WHIP homologue, MGS1 (maintenance of genome stability 1), can enhance the accelerated aging process in budding yeast. 34,45,46 The present investigation was undertaken to substantiate the interaction of WHIP with the NPC, followed by visualization of WHIP at the NE.…”