Interaction between the Rev1 C-Terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis

Abstract

Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι or Polκ, inserts a nucleotide across DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of 'inserter' to 'extender' DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the 'inserter' Polη, Polι or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit 'extender' Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits.

Translesion DNA polymerases in eukaryotes: what makes them tick?

“…These studies provide a molecular basis for a deeper insight into the highly complex mechanism mediating regulation and assembly of the TLS machinery. Thus, according to the current understanding, instead of competing for Rev1 binding, implied by the original model (Guo et al , 2003; Ohashi et al , 2004), pol ζ and one of the Y-family TLS polymerases cooperate in lesion bypass by simultaneous binding to the Rev1 scaffold (Kikuchi et al , 2012; Liu et al , 2013; Pozhidaeva et al , 2012; Pustovalova et al , 2012; Pustovalova et al , 2016; Wojtaszek et al , 2012a; Wojtaszek et al , 2012b; Xie et al , 2012; Xing et al , 2009) (Figure 3). This model suggests a mechanism by which Rev1 facilitates polymerase switching that is required to catalyze the two consecutive steps of TLS (Figure 3B & C) without the need for polymerase dissociation: 1) nucleotide insertion opposite the damaged site and 2) extension of the nascent strand past the lesion [for the origins of the two-step TLS model see (Bridges and Woodgate, 1985; Goodman and Woodgate, 2013; Livneh et al , 2010; Prakash and Prakash, 2002)].…”

“…For example, it has been reported that the large region of hREV7 (~130 amino acids) is required for the interaction with the C-terminal domain of hREV1 (Murakumo et al , 2001) and an approximately 100 amino acid region in C-terminal domain of human REV1 (Figure 2A, Protein Interaction Region, PIR) is required for the interaction with all other polymerases from the Y-family, as well as with Rev7 and PolD3 subunits of pol ζ (Figure 2C) (Guo et al , 2003; Masuda et al , 2003; Murakumo et al , 2001; Ohashi et al , 2004; Pustovalova et al , 2016). On the other hand, sequences as short as 16 amino acids in pols η, ι and κ (Figure 2B, one Rev1-interacting region (RIR) in pols ι and κ and two such motifs in pol η) are sufficient for recognition by hRev1 (Figure 2C) (Ohashi et al , 2009).…”

“…For example, it has been shown that there are two different interfaces within a 100 amino acid region in the C-terminal domain of human Rev1 (Figure 2A, PIR) (Kikuchi et al , 2012; Pustovalova et al , 2012). The C-terminal part of this domain (C) is required for the interaction with the Rev7 subunit of pol ζ, while the opposite surface (N-terminal part) of the CTD of Rev1 (N) is involved in an interaction with the short RIR motifs of pols η, ι and κ (Figure 2B, RIR) (Kikuchi et al , 2012; Pustovalova et al , 2012; Wojtaszek et al , 2012b), as well as of PolD3 subunit of pol ζ (Pustovalova et al , 2016). These studies provide a molecular basis for a deeper insight into the highly complex mechanism mediating regulation and assembly of the TLS machinery.…”

“…This model suggests a mechanism by which Rev1 facilitates polymerase switching that is required to catalyze the two consecutive steps of TLS (Figure 3B & C) without the need for polymerase dissociation: 1) nucleotide insertion opposite the damaged site and 2) extension of the nascent strand past the lesion [for the origins of the two-step TLS model see (Bridges and Woodgate, 1985; Goodman and Woodgate, 2013; Livneh et al , 2010; Prakash and Prakash, 2002)]. Conformational changes in REV7 induced by binding to PolD3 mediates the interaction with Rev1 (Hanafusa et al , 2010; Hara et al , 2010) thereby ensuring efficient assembly of the “extender” complex (multi-subunit pol ζ) and facilitating displacement of the “inserter” polymerase (one of the members of the Y-family) (Kikuchi et al , 2012; Pustovalova et al , 2016) (Figure 3C). Furthermore, Rev1 seems to be instrumental in the selection of the best-suited polymerase for each TLS step via coordination of the replacement of a suboptimal DNA polymerase with a more appropriate enzyme, i.e., the one which is able to complete the task by catalyzing TLS of a specific lesion most efficiently and accurately (Ohashi et al , 2009).…”

Identification of Small Molecule Translesion Synthesis Inhibitors That Target the Rev1-CT/RIR Protein−Protein Interaction

“…6,9 In humans, Rev1 PPIs with all other TLS enzymes are mediated by its C-terminal (Rev1-CT) domain that can bind R ev1- i nteracting r egions (RIR) from Polκ, Polη, Polι and PolD3 and, at the same time, interact with the accessory Rev7 subunit of Polζ. 29–35 Cells deficient in Rev1 exhibit increased sensitivity to DNA damage and a significantly reduced mutation rate. 36–38 Deletion of the Rev1-CT domain confers a similar DNA damage sensitive non-mutable phenotype, suggesting that this domain is critical for the cellular function of the TLS pathway.…”