Abstract
Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη 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 Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι 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 Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.
Highlights
Translesion DNA synthesis (TLS), a DNA damage tolerance mechanism, is a crucial biological function that protects cells from various genotoxic agents
Durando and co-workers reported that depletion of endogenous Pol decreases the levels of damage-induced mUb-proliferating cell nuclear antigen (PCNA), and ectopically expressed Pol promotes mono-ubiquitination of PCNA in cells in a manner that depends on PIP2 at the C-terminus [29]
To determine whether PIP1 plays any role in the promotion of PCNA mono-ubiquitination, we introduced the pip1 or pip2 mutation into FLAG-tagged Pol (Figure 1A), expressed the mutant proteins in xeroderma pigmentosum variant (XP-V) cells, and analysed the levels of mUb-PCNA in cells by western blotting
Summary
Translesion DNA synthesis (TLS), a DNA damage tolerance mechanism, is a crucial biological function that protects cells from various genotoxic agents. Each of the three Y-family DNA polymerases described above has one or two copies of the ubiquitin-binding domain (UBD), called UBZ (ubiquitinbinding zinc-finger) in Pol and Pol and UBM (ubiquitinbinding motif) in Pol [6]. These findings support the notion that mono-ubiquitination of PCNA plays a key role in switching from replicative DNA polymerase stalled at a site of DNA damage to a DNA polymerase (such as Pol, or ) capable of carrying out TLS [14,15,20,21]. This idea is still controversial [5], and more recent publications report that Pol and Pol are able to carry out TLS independently of PCNA ubiquitination in some circumstances [22,23,24]
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