Comparative analysis of in vivo interactions between Rev1 protein and other Y-family DNA polymerases in animals and yeasts
Introduction
The rescue of arrested DNA replication at sites of template base damage is critical for cell survival. Not surprisingly, prokaryotic and eukaryotic cells have evolved multiple strategies for mitigating the lethal effects of arrested DNA replication without prior removal of the offending DNA damage; so-called DNA damage tolerance [1]. The replicative bypass of base damage by DNA translesion synthesis (TLS) represents a specific mode of damage tolerance that utilizes specialized low-fidelity DNA polymerases to overcome arrested DNA replication, often at the expense of introducing errors and hence generating mutations [1]. To date ten such specialized DNA polymerases have been identified in vertebrates. A newly discovered subset of these proteins (Rev1, Polη, Polι, and Polκ) is designated the Y-family of DNA polymerases [1], [2], [3].
Among the Y-family of DNA polymerases Rev1 protein is highly conserved in eukaryotes, but no archaeal or bacterial Rev1 orthologs have been detected. Structural orthologs of Polη and Polι are also apparently absent in prokaryotes. In contrast, a readily identifiable ortholog of Polκ (DinB protein in E. coli) is present in bacteria. Rev1 is unique among the Y-family in that its DNA polymerase activity is restricted to the incorporation of one or two molecules of dCMP regardless of the nature of the template nucleotide. It is thus often referred to as a dCMP transferase [4]. Remarkably, while the catalytic domain of Rev1 protein is required for the replicative bypass of sites of base loss (AP sites), inactivation of this activity does not abrogate a requirement for Rev1 for ultraviolet (UV) radiation-induced mutagenesis in yeast or mammalian cells [5], [6], [7].
Rev1 protein also possesses a conserved N-terminal BRCT domain that is required for TLS in yeast and mammalian cells exposed to UV radiation [7], [8] and presumably other types of base damage. Indeed, a single amino acid substitution in the BRCT domain of otherwise catalytically active yeast Rev1 abolishes the bypass of [6-4] photoproducts, suggesting a non-catalytic role(s) for Rev1 protein during UV radiation-induced mutagenesis [7]. Additional support for the notion that Rev1 has a function(s) in TLS that is independent of its dCMP transferase activity is implicit in the observation that the protein interacts with the Y-family polymerases Polκ, Polη and Polι, and with Rev7 protein [a subunit of a heterodimeric specialized DNA polymerase called Polζ] through a C-terminal 100 amino acid region that is highly conserved among vertebrates [9], [10], [11]. The functional significance of these interactions is not understood. However, the additional observations that PCNA also interacts with these DNA polymerases and with Rev1 protein [8], [12], [13], [14], and that PCNA and some Y-family members (including Rev1 protein) undergo monoubiquitination, has prompted the hypothesis that Rev1 plays a key role in the process of TLS [3], [15], [16].
Several non-vertebrate eukaryotic organisms, such as the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the fruit fly Drosophila melanogaster, and the nematode Caenorhabditis elegans, have proven to be informative model systems for various mechanistic studies in vertebrates. In view of the fact that these model organisms are endowed with Rev1 protein as well as one or more other Y-family DNA polymerases, they offer the potential for gaining fundamental insights into the molecular biology of TLS in eukaryotes. In the present studies we have compared interactions between Rev1 protein and other members of the Y-family of DNA polymerases from animals and fungi.
Here we report that Rev1 protein from the fruit fly D. melanogaster and the yeasts S. cerevisiae and S. pombe readily interacts with the Rev7 subunit of the specialized DNA polymerase ζ (Polζ). Additionally, various Y-family DNA polymerases from Drosophila interact with Rev1 protein from this organism. In contrast, members of the Y-family of specialized DNA polymerases from both yeasts and from C. elegans do not interact with Rev1 protein from these organisms. Consistent with this observation, the extensive conservation of the C-terminal 100 amino acids of Rev1 protein in vertebrates is not observed in yeasts or nematodes. Remarkably, however, the extent of amino acid conservation in the C-terminal region of Rev1 protein from Drosophila is not obviously greater than that observed in yeast.
In contrast to the corresponding mouse proteins, the Drosophila Y-family DNA polymerases Polι and Polη utilize two distinct regions to interact with Drosophila Rev1. However, a comparison of the Rev1-interacting domains in Polη, Polι and Polκ from mouse or Drosophila reveals little sequence conservation and does not predict conserved structures. Thus, notwithstanding the presence of Rev1 protein and some specialized DNA polymerases in invertebrates and fungi, interactions between these proteins differ qualitatively among themselves and from the Rev1–DNA polymerase interactions observed in vertebrates. We conclude that no single eukaryotic model system thus far examined can be considered a prototypic model system for generalizing the molecular mechanism of TLS in eukaryotes, and suggest that care must be exercised in making mechanistic extrapolations from one eukaryotic system to another.
Section snippets
S. cerevisiae constructs
Rev1 was PCR amplified from Rev1p-GST-pJN60 [17] and cloned into pACT2 (Clontech) or pGBKT7 (Clontech). Rad30 was PCR amplified from pEGUh6b-Rad30 [18] and cloned into pGBKT7 or pGBT9 (Clontech). Rev7 was PCR amplified by colony PCR and cloned into pGADT7 (Clontech).
C. elegans constructs
Rev-1 was amplified by RT-PCR of total RNA (prepared by bead disruption and RNAeasy prep of N2 hermaphrodite worms) and cloned into pGADT7. Polη-1 was amplified by RT-PCR and cloned into pGBKT7. Two spliced products were detected,
Interactions between Rev1 protein and Rev7 protein, the catalytic subunit of the B-family DNA polymerase Polζ
In addition to its well-documented ability to interact with various Y-family DNA polymerases, the highly conserved C-terminal region of mouse Rev1 protein interacts with the Rev7 subunit of Polζ, a specialized DNA polymerase from the B-family, which is also implicated in TLS in eukaryotes. Rev1 protein from Drosophila and the yeasts S. pombe and S. cerevisiae also interacts with homologous Rev7 protein (Fig. 1, Fig. 2, Fig. 3, Fig. 4). Additionally, mouse Rev1 maintains an interaction with Rev7
Discussion
Previous studies indicate that Rev1 protein in eukaryotic organisms maintains one or more functions in DNA damage tolerance independent of its dCMP transferase activity [6], [7]. In light of the observation that human and mouse Rev1 interact with multiple Y-family DNA polymerases via a highly conserved C-terminal domain [9], [10], [11], we inquired whether similar if not identical interactions are conserved in invertebrates and fungi that also possess Y-family homologues. Surprisingly, given
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We are grateful to Dr. Timothy Megraw and Shaila Kotadia for providing materials and protocols for Drosophila experiments. We thank Christopher Lawrence, Zhigang Wang, and Takeshi Todo for providing polymerase constructs. We also thank Burke Squires for assistance with preliminary sequence alignments and acknowledge the C. elegans Genome Consortium for providing N2 hermaphrodite worms. G.C.W. is an American Cancer Society Research Professor and is supported by National Cancer Institute (NCI)
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2015, DNA RepairCitation Excerpt :This regulatory role of Rev1 strongly relies on (i) an N-terminal functional unit consisting of the BRCT domain with preceding α helices [Fig. 1], [10,47–51], (ii) ubiquitin-binding motifs and (iii) the C-terminus of Rev1 (Fig. 1) which, at least in vertebrates, binds the other Y-family polymerases [52–55] and Rev7 [54,56–60]. It is noteworthy that in contrast to vertebrate Rev1, S. cerevisiae Rev1 binds to Pol η at its polymerase-associated domain (PAD), which is located upstream from Rev1's C terminus [Fig. 1], [61,62]. It is still not clear how defects in Pol ζ or in Rev1, including mutations in the BRCT domain, can lead to hypomutability.
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2015, DNA RepairCitation Excerpt :One group reported an interaction between the little finger domain of Rev1 and Pol η [95]. However, another group failed to detect an interaction between these two yeast proteins while the interaction between the mammalian orthologs was readily observable [93]. Mutagenesis is regulated at several levels, including by post-translational modifications (Table 2).
NMR mapping of PCNA interaction with translesion synthesis DNA polymerase Rev1 mediated by Rev1-BRCT domain
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