Linkage reprogramming by tailor-made E3s reveals polyubiquitin chain requirements in DNA-damage bypass.

A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo can change the fate of the modified substrate. We also provide evidence for redundancy between distinct but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates a promising approach toward the in vivo analysis of polyubiquitin signaling. [Display omitted] • Tailor-made E3s afford PCNA polyubiquitylation with alternative linkages • Alternative linkages confer distinct fates to polyubiquitylated PCNA • Linkage reprogramming is generalizable to other monoubiquitylated target proteins Wegmann et al. have designed tailor-made ubiquitin protein ligases for substrate-selective extension of polyubiquitin chains with defined linkages. Application to the replication factor PCNA shows that alternative linkages confer distinct fates to the modified target. Proof-of-principle experiments demonstrate that the method of linkage reprogramming is generalizable to other monoubiquitylated proteins. [ABSTRACT FROM AUTHOR]