Complex Formation of Yeast Rev1 and Rev7 Proteins: a Novel Role for the Polymerase-Associated Domain

Abstract

The Rev1 protein of Saccharomyces cerevisiae functions in translesion synthesis (TLS) together with DNA polymerase (Pol) , which is comprised of the Rev3 catalytic and the Rev7 accessory subunits. Rev1, a member of the Y family of Pols, differs from other members in its high degree of specificity for incorporating a C opposite template G as well as opposite an abasic site. Although Rev1 is indispensable for Pol-dependent TLS, its DNA synthetic activity is not required for many of the Pol-dependent lesion bypass events. This observation has suggested a structural role for Rev1 in this process. Here we show that in yeast, Rev1 forms a stable complex with Rev7, and the two proteins copurify. Importantly, the polymerase-associated domain (PAD) of Rev1 mediates its binding to Rev7. These observations reveal a novel role for the PAD region of Rev1 in protein-protein interactions, and they raise the possibility of a similar involvement of the PAD of other Y family Pols in protein-protein interactions. We discuss the possible roles of Rev1 versus the Rev1-Rev7 complex in TLS.Rad6, a ubiquitin-conjugating enzyme, exists in vivo in a tight complex with Rad18, which is a DNA binding protein (1, 2) and also acts as an E3 in the ubiquitin conjugation process (11). Rad6-Rad18-mediated ubiquitin conjugation promotes replication through DNA lesions via at least three different pathways: DNA polymerase (Pol) -dependent translesion synthesis (TLS), Pol dependent TLS, and a Rad5-Mms2-Ubc13-dependent pathway, the mechanism of which is not known (41).Pol promotes efficient and relatively error-free synthesis through UV-induced cyclobutane pyrimidine dimers which form at TT, TC, and CC sites (12,16,45,46); consequently, inactivation of Pol in Saccharomyces cerevisiae and human cells confers enhanced UV mutagenesis (13,30,39,44,47,48) and in humans causes the variant form of xeroderma pigmentosum (12,29). Pol, which is comprised of the Rev3 catalytic subunit and the Rev7 accessory subunit (33), is indispensable for UV mutagenesis in yeast (19,21,23) as well as human cells (6,18,24), and genetic studies in yeast have indicated its requirement for mutagenesis resulting from TLS occurring through abasic sites (14) and also through bases damaged upon treatment with certain chemical agents (35). Pol promotes lesion bypass primarily via its role as an extender, wherein following the insertion of a nucleotide opposite the DNA lesion by an inserter polymerase, Pol performs the extension of the nascent primer terminus (9, 15, 17, 37). For its role in TLS, however, Pol requires the Rev1 protein, which like the Rev3 and Rev7 proteins is indispensable for UV mutagenesis (19,20,22,23,36) and for mutagenesis resulting from TLS occurring through abasic sites (14) and through other damaged bases (3).Rev1, although a member of the Y family of DNA polymerases, differs from the other members in its specificity for predominantly inserting a C opposite template G and also opposite the other template nucleotides as well as an abasic site. Thus, Rev1 is a h...

Complex Formation of Yeast Rev1 with DNA Polymerase η

“…In yeast, Rev1 also binds to the Rev3 subunit of Pol and this interaction is inhibited in Rev1 with the C-terminal 72 residues deleted (2). Yeast Rev1 has also been shown to bind the Rev7 protein, and this interaction requires the same PAD region of Rev1 as is involved in Pol binding (1). The physical interaction of Rev1 with Pol does not occur in the Rev1-Rev7 complex.…”

“…The physical interactions between Pol and Rev1 proteins were carried out by using a protocol described before (1,2). Briefly, GST-Rev1 or its truncated forms were incubated with Pol or its truncated derivatives and vice versa in buffer I (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 5 mM dithiothreitol [DTT], 0.01% NP-40, and 10% glycerol) in a 20-l reaction mixture at 4°C for 30 min, followed by 10 min at 25°C.…”

“…To obtain untagged proteins, GST-fused proteins bound to glutathione-Sepharose beads were treated overnight at 4°C with PreScission protease to cleave between the GST tag and Pol or Rev1. For purification of the Rev1-Rev7 complex, the GST-Rev1-Rev7 complex was purified from yeast cells carrying the plasmids for expressing the GST-Rev1 and Rev7 proteins and the GST tag was cleaved from Rev1 as previously described (1).…”

“…Rev7 is an accessory but essential subunit of Pol. Rev7 has also been shown to bind directly to Rev1, and a stable Rev1-Rev7 complex can be purified from yeast cells (1). Previously, we have shown that unlike Rev1, the Rev1-Rev7 complex does not bind Rev3; thus, the presence of Rev7 in the Rev1-Rev7 complex is inhibitory to the physical interaction of Rev1 with Rev3 in the Pol (Rev3-Rev7) complex (2).…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…The yeast rev1 mutant displays a complete loss of mutagenesis activity comparable to that of rev3, which cannot be explained by its dCMP transferase activity. Indeed, analysis of site-specific mutations confirms that the Rev1 enzymatic activity is not essential for TLS, but its BRCA1 C-terminal (BRCT) domain [94,95] and/or a polymerase-associated domain (PAD) [96] are required for protein interactions. The C-terminal 100 amino acids of human Rev1 are sufficient to interact with all other TLS polymerases [97] ( Figure 3), implying a scaffold role of Rev1 in TLS.…”