The Non-canonical Protein Binding Site at the Monomer-Monomer Interface of Yeast Proliferating Cell Nuclear Antigen (PCNA) Regulates the Rev1-PCNA Interaction and Polζ/Rev1-dependent Translesion DNA Synthesis

Abstract

Rev1 and DNA polymerase (Pol) are involved in the tolerance of DNA damage by translesion synthesis (TLS). The proliferating cell nuclear antigen (PCNA), the auxiliary factor of nuclear DNA polymerases, plays an important role in regulating the access of TLS polymerases to the primer terminus. Both Rev1 and Pol lack the conserved hydrophobic motif that is used by many proteins for the interaction with PCNA at its interdomain connector loop. We have previously reported that the interaction of yeast Pol with PCNA occurs at an unusual site near the monomer-monomer interface of the trimeric PCNA. Using GST pull-down assays, PCNA-coupled affinity beads pulldown and gel filtration chromatography, we show that the same region is required for the physical interaction of PCNA with the polymerase-associated domain (PAD) of Rev1. The interaction is disrupted by the pol30-113 mutation that results in a double amino acid substitution at the monomer-monomer interface of PCNA. Genetic analysis of the epistatic relationship of the pol30-113 mutation with an array of DNA repair and damage tolerance mutations indicated that PCNA-113 is specifically defective in the Rev1/Pol-dependent TLS pathway. Taken together, the data suggest that Pol and Rev1 are unique among PCNA-interacting proteins in using the novel binding site near the intermolecular interface of PCNA. The new mode of Rev1-PCNA binding described here suggests a mechanism by which Rev1 adopts a catalytically inactive configuration at the replication fork.Cellular DNA is continuously attacked by endogenous and exogenous agents that damage the bases and the DNA backbone. Damage that is not repaired prior to the S phase of cell cycle can block the replication machinery, because most lesions cannot be accommodated in the highly selective active site of replicative DNA polymerases (1, 2). Replication stalling activates several damage tolerance mechanisms that help bypass the damage in either an accurate or mutagenic manner. Translesion DNA synthesis (TLS) 3 is an important damage tolerance pathway, in which specialized DNA polymerases are recruited to synthesize DNA through template lesions (3). Structural studies showed that TLS polymerases have a more open active site that allows them to accommodate a variety of DNA lesions and catalyze polymerization on damaged templates (4). The accuracy of TLS can vary depending on the particular lesion and the DNA polymerases involved. It is, however, an inherently mutagenic process, because the damage can alter the coding properties of the bases, and because TLS polymerases generally have low fidelity (2).In human cells, TLS polymerases include Y family enzymes Pol, Pol, Pol, and REV1, and the B family enzyme Pol. Although the Y family polymerases share little amino acid sequence similarity with the classical DNA polymerases, they have a similar overall "right-hand" architecture with the "palm," "thumb," and "fingers" domains. The thumb and fingers domains of the Y family enzymes, however, are short and do not make as many contacts wi...

The Vital Role of Polymerase ζ and REV1 in Mutagenic, but Not Correct, DNA Synthesis across Benzo[a]pyrene-dG and Recruitment of Polymerase ζ by REV1 to Replication-stalled Site

“…However, de Groote et al (44) recently argued against this assertion by showing the BRCT domain, together with a further N-terminal region, to bind to recessed, phosphorylated 5Ј ends of double-stranded DNA. REV1 probably has a PCNA-binding site(s) in its C-terminal region (12,46,47). Furthermore, although Akagi et al (48) have shown that the accumulation of human endogenous REV1 into locally UV-irradiated regions of nucleus depends on the interaction with pol , Andersen et al (45) have reported that the recruitment of the human endogenous REV1 is independent of the interaction with pol .…”

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link, Enhances DNA Double Strand Break, and Sensitizes Cancer Cells to Cisplatin

“…It could be due to different affinities of pol and REV1 for the monoubiquitinated PCNA homotrimer. Yeast REV1 reportedly binds to PCNA on the homotrimer region by its PAD domain (38). If this interaction is also valid in human REV1, a small molecule that binds to the PCNA homotrimer region, such as T2AA, could efficiently inhibit REV1 functions.…”

Eukaryotic DNA polymerase ζ

“…Instead, mono-ubiquitination of PCNA likely exerts its TLS-promoting activity through Rev1. Rev1 interacts with PCNA through its BRCT-domain and through the polymerase-associated domain [106,108,109], and shows additional interactions with ubiquitinated PCNA through its UMB motif [107,110]. The affinity of Rev1 for mono-ubiquitinated PCNA is higher than that for unmodified PCNA, further emphasizing the essential role of Rev1 in mutagenesis [107,110,111].…”