Comparative analysis of in vivo interactions between Rev1 protein and other Y-family DNA polymerases in animals and yeasts

Abstract

SummaryEukaryotes are endowed with multiple specialized DNA polymerases, some (if not all) of which are believed to play important roles in the tolerance of base damage during DNA replication. Among these DNA polymerases, Rev1 protein (a deoxycytidyl transferase) from vertebrates interacts with several other specialized polymerases via a highly conserved C-terminal region. The present studies assessed whether these interactions are retained in more experimentally tractable model systems, including yeasts, flies, and the nematode C. elegans. We observed a physical interaction between Rev1 protein and other Y-family polymerases in the fruit fly Drosophila melanogaster. However, despite the fact that the C-terminal region of Drosophila and yeast Rev1 are conserved from vertebrates to a similar extent, such interactions were not observed in S. cerevisiae or S. pombe. With respect to regions in specialized DNA polymerases that are required for interaction with Rev1, we find predicted disorder to be an underlying structural commonality. The results of this study suggest that special consideration should be exercised when making mechanistic extrapolations regarding translesion DNA synthesis from one eukaryotic system to another.

Structural Basis of Rev1-mediated Assembly of a Quaternary Vertebrate Translesion Polymerase Complex Consisting of Rev1, Heterodimeric Polymerase (Pol) ζ, and Pol κ

“…The final crystallization sample containing the quaternary complex of the Rev1 CTD, Rev3/7, and Pol RIR complex was prepared by passing the protein mixture of Rev3/7 with a slight molar excess amount of the Rev1 CTD-Pol RIR complex through a size exclusion column ( 15 N-labeled Rev1 CTD in the presence of equal molar ratios of both GB1-Pol RIR and Pol was also recorded to establish the formation of the quaternary Rev1 CTD-Rev3/ 7-Pol RIR complex.…”

“…Rev1 interaction is required for the protective role of Pol against benzo[a]pyrene in mammalian cells, and it promotes Pol -mediated suppression of spontaneous mutations in human cells (17,18). Although the interactions between the Rev1 CTD and the RIRs of Y-family polymerases , , and are only found in vertebrates (15), the Rev1 CTD-Pol interaction is evolutionarily conserved from yeast to humans, highlighting the essential role of the Rev1-Pol interaction in translesion synthesis. Consistent with this notion, the Rev1 CTD is required for effective DNA damage tolerance in mammalian cells, and mutations of the Rev1 CTD display a profound defect in translesion synthesis with significantly reduced damage-induced mutagenesis in yeast (11,19).…”

“…The scaffolding function of Rev1 in vertebrates is critically dependent on its C-terminal domain of ϳ100 amino acids (11), which has been shown to bind insertion polymerases , , and through their Rev1-interacting region (RIR) (12)(13)(14)(15) and extension polymerase through its Rev7 subunit (12,13,16). Rev1 interaction is required for the protective role of Pol against benzo[a]pyrene in mammalian cells, and it promotes Pol -mediated suppression of spontaneous mutations in human cells (17,18).…”

The DNA Polymerase Activity ofSaccharomyces cerevisiaeRev1 is Biologically Significant

“…Mutants in the UBM2 of Rev1 also display decreased survival and mutagenesis in response to DNA damage that is likely caused by a less efficient interaction with monoubiquitinated PCNA (Guo et al 2006b;Wood et al 2007;D'Souza et al 2008;Bomar et al 2010). Additional studies found that the last $100 amino acids of Rev1 could interact with the other TLS polymerases in mammalian cells (Murakumo et al 2001;Guo et al 2003;Ohashi et al 2004;Tissier et al 2004) and in S. cerevisiae (Acharya et al 2005(Acharya et al , 2006D'Souza and Walker 2006;D'Souza et al 2008;Kosarek et al 2008). Not surprisingly, truncations of, or point mutations in, the C terminus cause severe survival and mutagenesis defects after DNA damage (Larimer et al 1989;Ross et al 2005;Acharya et al 2006;D'Souza et al 2008;Kosarek et al 2008).…”

“…Additional studies found that the last $100 amino acids of Rev1 could interact with the other TLS polymerases in mammalian cells (Murakumo et al 2001;Guo et al 2003;Ohashi et al 2004;Tissier et al 2004) and in S. cerevisiae (Acharya et al 2005(Acharya et al , 2006D'Souza and Walker 2006;D'Souza et al 2008;Kosarek et al 2008). Not surprisingly, truncations of, or point mutations in, the C terminus cause severe survival and mutagenesis defects after DNA damage (Larimer et al 1989;Ross et al 2005;Acharya et al 2006;D'Souza et al 2008;Kosarek et al 2008). Given that these domains bind other proteins and contribute to survival and mutagenesis for a spectrum of DNA damage, it has been proposed that Rev1 acts a scaffolding protein that promotes the recruitment of TLS polymerases to sites of DNA adducts and/or aids in the recognition of DNA lesions (Waters et al 2009).…”