LIG_REV1ctd_RIR_1

The RIR motif plays a role in linking DNA repair proteins. When the replication fork encounters a DNA lesion (a persisting small distortion in DNA), a high-fidelity replicative DNA polymerase (such as Polδ or Polη) with proof-reading 3′–5′ exonuclease activity often stalls at the damaged site. For replication to be continued, the stalled replicative DNA polymerase must be replaced with a DNA polymerase that is capable of carrying out translesion DNA synthesis (TLS). Rev1/Polζ-dependent TLS is performed in a two-step process. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ (Ohmori,2001; Ohashi,2009) inserts a nucleotide across a DNA lesion. In the second step, the four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/Polδ2/Polδ3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the sliding clamp PCNA and the modular TLS polymerase Rev1, which is a major scaffold for TLS that is also capable of catalysing deoxycytidyl transfer to the 3’ end of a DNA primer. Y-family polymerases need to interact with REV1 for TLS at a stage subsequent to their PCNA-dependent recruitment to stalled replication forks (Ohashi,2009). Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CTD), which can simultaneously bind the Rev7 subunit of Polζ on one surface and on the other side, Rev1-interacting region (RIR) motifs from Polη, Polι, Polκ or XRCC1 (Liu,2013; Pustovalova,2016). Since the Rev1-CTD utilizes independent interaction interfaces to bind a fragment of the “inserter” Y-family polymerase and Rev7 subunit of the “extender” Polζ, it serves as a scaffold that may accommodate several polymerases making them instantaneously available for TLS (4FJO; 4GK5; Pustovalova,2012; Wojtaszek,2012; Xie,2012; Liu,2013). Also, Rev1-CTD can bind the RIR of the Polζ subunit, Polδ3, with relatively high affinity (Pustovalova,2016). This supports a model of reorganization of the multipolymerase complex between the two steps of Rev1/Polζ-dependent TLS. The assembly of the “extender” four-subunit Polζ results in Polζ acquiring Rev1 and PCNA interacting motifs found in the Polδ3 subunit that help Polζ to displace the “inserter” Y-family enzyme via an affinity driven competition (Pustovalova,2016). Thus, besides its catalytic activity, Rev1 probably serves as a major platform orchestrating polymerase exchange steps during TLS.
Based on the validated RIR motifs of Y-family polymerases (Polκ 2LSI, 2LSJ; Polη 2LSK; Polδ3 2N1G) and other DNA repair proteins (e.g. XRCC1 and Spartan) RIRs bind a hydrophobic cavity of the Rev1-CTD four-helix bundle while folding up into variably long alpha-helices (Pozhidaeva,2012; Wojtaszek,2012; Pustovalova,2012). RIRs consist of two Phe residues facilitating the majority of contacts (Pustovalova,2012) followed by helix-forming residues that preferentially involve one or more basic residues to interact with the acidic surface of Rev1 (4FJO; Wojtaszek,2012; Liu,2013).
The RIR motif-mediated interaction with Rev1-CTD is conserved from yeasts to mammals, since the yeast proteins Polη and Msh6 (Boehm,2016) also employ RIRs to bind yeast Rev1-CTD.
The aromatic residue pair and basic amino acid preference of RIRs is quite similar to PIP Box and MIP box and so these motifs are thought to be able to overlap within a sequence (Boehm,2016; Boehm,2016).