Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases

Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Pol eta, Pol iota and Pol kappa, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Pol eta has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Pol eta foci, which co-localize with PCNA. We also show that Pol kappa has two functionally distinct PIP boxes, like Pol eta, whereas Pol iota has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Pol eta exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.