The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
RIR motif
Functional site description:
The replicative bypass of DNA lesions is performed in a two-step process, termed translesion synthesis (TLS). First, a Y-family TLS DNA polymerase, typically Pol eta (Polη), Pol iota (Polι), or Pol kappa (Polκ), inserts a nucleotide across the DNA lesion. Then, the Pol delta-containing Pol zeta complex (Polζ, a four-subunit Rev3/Rev7/Polδ2/Polδ3 B-family DNA polymerase) extends the distorted DNA primer template. The coordinated action of these TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 is an unusual Y-family polymerase with deoxycytidyl transferase activity and protein template-directed nucleotide incorporation. In a second function, Rev1 also acts as a major scaffold for TLS that orchestrates the exchange of different polymerases. The Rev1 C-terminal domain (Rev1-CTD) can simultaneously bind the Rev7 subunit of Polζ and the Rev1-interacting regions (RIRs) from Polη, Polι, Polκ (4FJO), Polδ3 or XRCC1.
ELM Description:
The RIR motif has two strictly conserved phenylalanine residues with the remaining positions rather loosely defined (Ohashi,2009). There are several structures of RIR motifs of Y-family polymerases binding to the Rev1-CTD (Polκ 2LSI; Polη 2LSK; Polδ3 2N1G). The Rev1-CTD is a four-helix bundle domain. Six residues at the N-terminus of the CTD α1 helix fold into a structurally defined β-hairpin, and together with the hydrophobic surface between α1 and α2, create a deep hydrophobic cavity (Pozhidaeva,2012; 22700976). Initially disordered RIR peptides fold into variable length α-helices on binding (Pustovalova,2016), with the two core Phe residues deeply inserted into the hydrophobic cavity of Rev1-CTD (Zhao,2017). The Phe residues are in the first turn of an α helix and their peptide backbone NH moieties H-bond to Asp1184 in Rev1, thereby positioning the Phe sidechains.
Several studies attempted to define the RIR motif pattern based on deletion, alanine scanning (Ohashi,2009), structural (Pustovalova,2012) and conservation data (Wojtaszek,2012). The two Phes provide the core of the motif, mediating most of the side chain contacts with the Rev1-CTD. In the position directly preceding the first Phe, helix N-capping residues are preferred, especially Ser, Asn and Pro (Pustovalova,2012), although not strictly required, because mutating this residue to Ala in Polκ did not eliminate binding (Ohashi,2009). In the four helical residue positions minimally required to follow the two Phes helix-breaking Pro residues are not allowed (Ohashi,2009). In most known RIRs, conserved positively charged residues occur in the 2nd or 3rd position (often both) following the FF core, mediating shallow electrostatic interactions with the acidic surface of Rev1 (4FJO; 2LSJ; Wojtaszek,2012). However, changing them to alanines did not have a significant impact on binding affinity according to some studies (Ohashi,2009; Gabel,2013).
Pattern: ..FF[^P]{0,2}[KR]{1,2}[^P]{0,4}
Pattern Probability: 0.0005350
Present in taxon: Eukaryota
Interaction Domain:
REV1_C (PF16727) DNA repair protein REV1 C-terminal domain (Stochiometry: 1 : 1)
o See 10 Instances for LIG_REV1ctd_RIR_1
o Abstract
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).
o 13 selected references:

o 7 GO-Terms:

o 10 Instances for LIG_REV1ctd_RIR_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q03834 MSH6
31 38 MKQSSLLSFFSKQVPSGTPS TP 4 Saccharomyces cerevisiae (Baker"s yeast)
Q04049 RAD30
625 632 PQKKQVTSSKNILSFFTRKK TP 6 Saccharomyces cerevisiae S288c
Q60596 Xrcc1
191 200 SLKPGALFFSRINKTSSAST TP 3 Mus musculus (House mouse)
418 428 KTVFDNFFIKKEQIKSSGND TP 1 Homo sapiens (Human)
Q15054 POLD3
236 245 KGNMMSNFFGKAAMNKFKVN TP 3 Homo sapiens (Human)
569 579 SRGVLSFFSKKQMQDIPINP TP 5 Homo sapiens (Human)
529 539 STGTEPFFKQKSLLLKQKQL TP 8 Homo sapiens (Human)
481 490 ATTSLESFFQKAAERQKVKE TP 5 Homo sapiens (Human)
Q9QUG2 Polk
564 575 MSHKKSFFDKKRSERISNCQ TP 2 Mus musculus (House mouse)
565 576 MSHKKSFFDKKRSERKWSHQ TP 17 Homo sapiens (Human)
Please cite: The eukaryotic linear motif resource - 2018 update. (PMID:29136216)

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