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 canchange the fate of the modified substrate. We also provide evidence for redundancy betweendistinct, 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 apromising approach towards the in vivo analysis of polyubiquitin signaling.