The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase η Mediates Its Interaction with the C-terminal Domain of Rev1

Abstract

Y-family DNA polymerases, such as polymerase , polymerase , and polymerase , catalyze the bypass of DNA damage during translesion synthesis. These enzymes are recruited to sites of DNA damage by interacting with the essential replication accessory protein proliferating cell nuclear antigen (PCNA) and the scaffold protein Rev1. In most Y-family polymerases, these interactions are mediated by one or more conserved PCNA-interacting protein (PIP) motifs that bind in a hydrophobic pocket on the front side of PCNA as well as by conserved Rev1-interacting region (RIR) motifs that bind in a hydrophobic pocket on the C-terminal domain of Rev1. Yeast polymerase , a prototypical translesion synthesis polymerase, binds both PCNA and Rev1. It possesses a single PIP motif but not an RIR motif. Here we show that the PIP motif of yeast polymerase mediates its interactions both with PCNA and with Rev1. Moreover, the PIP motif of polymerase binds in the hydrophobic pocket on the Rev1 C-terminal domain. We also show that the RIR motif of human polymerase and the PIP motif of yeast Msh6 bind both PCNA and Rev1. Overall, these findings demonstrate that PIP motifs and RIR motifs have overlapping specificities and can interact with both PCNA and Rev1 in structurally similar ways. These findings also suggest that PIP motifs are a more versatile protein interaction motif than previously believed. Proliferating cell nuclear antigen (PCNA)2 is an essential protein that functions in DNA replication, repair, recombination, damage tolerance, and cell cycle control. It is a homotrimer that forms a ring-shaped clamp, which slides along DNA (1-3). Many proteins involved in DNA metabolism, the maintenance of genome stability, and cell cycle control possess PCNA-interacting protein (PIP) motifs that mediate their binding to PCNA (1, 4 -6). These PIP motifs contain two adjacent aromatic residues, which bind in a hydrophobic pocket on the front face of the PCNA ring (2, 4), and the binding of these PIP motifs to PCNA represents a major point of regulation for these proteins.Among the proteins that are regulated by PCNA are the Y-family DNA polymerases such as DNA polymerase (pol ), DNA polymerase (pol ), DNA polymerase (pol ), and Rev1 (7-12). These polymerases bypass DNA damage during translesion DNA synthesis in order to allow for continuous progression of the replication fork (13-16). Pol , for example, catalyzes the bypass of UV-induced thymine-thymine dimers and 8-oxoguanine lesions (17, 18). Defects in pol cause the variant form of xeroderma pigmentosum, a genetic disorder characterized by sensitivity to sunlight and a high incidence of skin cancers (19,20). Rev1, by contrast, bypasses damaged guanines and abasic sites (21-24).In addition to its catalytic function, Rev1 plays an important non-catalytic role in translesion synthesis by binding to other polymerases, like pol , pol , and pol , and acting as a scaffold to recruit them to PCNA (25-29). Many Y-family polymerases, such as mammalian pol , pol , and pol , possess short, conserved Rev1...

Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases

“…One possible limitation of ITC is that is difficult to obtain a strong signal when the ΔH of binding is very small. Nevertheless, ITC has been used to examine the interactions between the PIP motif of pol η and both PCNA and the Rev1 CTD (Boehm et al, 2016a). It has also been used to study the interactions between the UBZ motif of human pol η and ubiquitin (Bomar et al, 2007).…”

“…Pol η has a single PIP motif in its disordered C-terminal region. This motif mediates interactions with PCNA by binding to a hydrophobic pocket on the front face of the PCNA ring (Figure 3A) (Boehm et al, 2016a; Haracska et al, 2001a). The pol η PIP motif also mediates interactions with Rev1 by binding to a hydrophobic pocket on the Rev1 CTD (Boehm et al, 2016a).…”

“…This motif mediates interactions with PCNA by binding to a hydrophobic pocket on the front face of the PCNA ring (Figure 3A) (Boehm et al, 2016a; Haracska et al, 2001a). The pol η PIP motif also mediates interactions with Rev1 by binding to a hydrophobic pocket on the Rev1 CTD (Boehm et al, 2016a). Given that the interactions of the pol η PIP motif with PCNA and with the Rev1 CTD are mutually exclusive, the ternary complexes formed by these proteins can only have two possible architectures: PCNA tool belts and Rev1 bridges (Figure 3B).…”

“…Single-molecule TIRF microscopy assays were used to study the interactions between pol η and PCNA as well as pol η and Rev1 (Boehm et al, 2016a). These studies showed that the K d for the pol η-PCNA and the pol η-Rev1 binary complexes have similar affinities.…”

Purification and optimization of conditions for DNA polymerase isolated from thermophile bacteria Bacillus caldolyticus

“…They have a crucial role in DNA replication, repair and recombination processes [2,3]. Two unclear phenomena in biology, cell proliferation and the impossibility of nerve cell division, are closely related to DNA polymerase activity [4]. Interest in thermophilic bacteria has been focused on the discovery of new strains, which can be used as a source for the production of thermostable enzymes [1].…”