A stapled POL κ peptide targets REV1 to inhibit mutagenic translesion synthesis.

Stapled α-helical RIR (Rev1-interacting region) peptides of DNA POL κ bind more effectively to the RIR-interface of the C-terminal recruitment domain of the translesion synthesis DNA polymerase Rev1 than unstapled peptide. The tightest-binding stapled peptide translocates into cells and enhances the cytotoxicity of DNA damaging agents while reducing mutagenesis. Drugs with these characteristics could potentially serve as adjuvants to improve chemotherapy and reduce acquired resistance by inhibiting Rev1-dependent mutagenic translesion synthesis.

Rev7 dimerization is important for assembly and function of the Rev1/Polζ translesion synthesis complex.

The translesion synthesis (TLS) polymerases Polζ and Rev1 form a complex that enables replication of damaged DNA. The Rev7 subunit of Polζ, which is a multifaceted HORMA (Hop1, Rev7, Mad2) protein with roles in TLS, DNA repair, and cell-cycle control, facilitates assembly of this complex by binding Rev1 and the catalytic subunit of Polζ, Rev3. Rev7 interacts with Rev3 by a mechanism conserved among HORMA proteins, whereby an open-to-closed transition locks the ligand underneath the "safety belt" loop. Dimerization of HORMA proteins promotes binding and release of this ligand, as exemplified by the Rev7 homolog, Mad2. Here, we investigate the dimerization of Rev7 when bound to the two Rev7-binding motifs (RBMs) in Rev3 by combining in vitro analyses of Rev7 structure and interactions with a functional assay in a Rev7-/- cell line. We demonstrate that Rev7 uses the conventional HORMA dimerization interface both to form a homodimer when tethered by the two RBMs in Rev3 and to heterodimerize with other HORMA domains, Mad2 and p31comet Structurally, the Rev7 dimer can bind only one copy of Rev1, revealing an unexpected Rev1/Polζ architecture. In cells, mutation of the Rev7 dimer interface increases sensitivity to DNA damage. These results provide insights into the structure of the Rev1/Polζ TLS assembly and highlight the function of Rev7 homo- and heterodimerization.

Interaction between the Rev1 C-Terminal Domain and the PolD3 Subunit of Polζ Suggests a Mechanism of Polymerase Exchange upon Rev1/Polζ-Dependent Translesion Synthesis.

Translesion synthesis (TLS) is a mutagenic branch of cellular DNA damage tolerance that enables bypass replication over DNA lesions carried out by specialized low-fidelity DNA polymerases. The replicative bypass of most types of DNA damage is performed in a two-step process of Rev1/Polζ-dependent TLS. In the first step, a Y-family TLS enzyme, typically Polη, Polι, or Polκ, inserts a nucleotide across a DNA lesion. In the second step, a four-subunit B-family DNA polymerase Polζ (Rev3/Rev7/PolD2/PolD3 complex) extends the distorted DNA primer-template. The coordinated action of error-prone TLS enzymes is regulated through their interactions with the two scaffold proteins, the sliding clamp PCNA and the TLS polymerase Rev1. Rev1 interactions with all other TLS enzymes are mediated by its C-terminal domain (Rev1-CT), which can simultaneously bind the Rev7 subunit of Polζ and Rev1-interacting regions (RIRs) from Polη, Polι, or Polκ. In this work, we identified a previously unknown RIR motif in the C-terminal part of PolD3 subunit of Polζ whose interaction with the Rev1-CT is among the tightest mediated by RIR motifs. Three-dimensional structure of the Rev1-CT/PolD3-RIR complex determined by NMR spectroscopy revealed a structural basis for the relatively high affinity of this interaction. The unexpected discovery of PolD3-RIR motif suggests a mechanism of "inserter" to "extender" DNA polymerase switch upon Rev1/Polζ-dependent TLS, in which the PolD3-RIR binding to the Rev1-CT (i) helps displace the "inserter" Polη, Polι, or Polκ from its complex with Rev1, and (ii) facilitates assembly of the four-subunit "extender" Polζ through simultaneous interaction of Rev1-CT with Rev7 and PolD3 subunits.

Characterization of a novel pyranopyridine inhibitor of the AcrAB efflux pump of Escherichia coli.

Members of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC of Escherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of the Enterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versus E. coli AB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 µM) in combination with 0.016 µg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) of E. coli AB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 µg/ml CIP alone. In contrast, phenyl-arginine-ß-naphthylamide (PAßN), a known EPI, did not increase the bactericidal activity of 0.016 µg/ml CIP at concentrations as high as 100 µM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active against Enterobacteriaceae species and Pseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.

Structural basis of Rev1-mediated assembly of a quaternary vertebrate translesion polymerase complex consisting of Rev1, heterodimeric polymerase (Pol) ζ, and Pol κ.

DNA synthesis across lesions during genomic replication requires concerted actions of specialized DNA polymerases in a potentially mutagenic process known as translesion synthesis. Current models suggest that translesion synthesis in mammalian cells is achieved in two sequential steps, with a Y-family DNA polymerase (κ, η, ι, or Rev1) inserting a nucleotide opposite the lesion and with the heterodimeric B-family polymerase ζ, consisting of the catalytic Rev3 subunit and the accessory Rev7 subunit, replacing the insertion polymerase to carry out primer extension past the lesion. Effective translesion synthesis in vertebrates requires the scaffolding function of the C-terminal domain (CTD) of Rev1 that interacts with the Rev1-interacting region of polymerases κ, η, and ι and with the Rev7 subunit of polymerase ζ. We report the purification and structure determination of a quaternary translesion polymerase complex consisting of the Rev1 CTD, the heterodimeric Pol ζ complex, and the Pol κ Rev1-interacting region. Yeast two-hybrid assays were employed to identify important interface residues of the translesion polymerase complex. The structural elucidation of such a quaternary translesion polymerase complex encompassing both insertion and extension polymerases bridged by the Rev1 CTD provides the first molecular explanation of the essential scaffolding function of Rev1 and highlights the Rev1 CTD as a promising target for developing novel cancer therapeutics to suppress translesion synthesis. Our studies support the notion that vertebrate insertion and extension polymerases could structurally cooperate within a megatranslesion polymerase complex (translesionsome) nucleated by Rev1 to achieve efficient lesion bypass without incurring an additional switching mechanism.

NMR structure and dynamics of the C-terminal domain from human Rev1 and its complex with Rev1 interacting region of DNA polymerase η.

Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal ß-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit µs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.

Multifaceted recognition of vertebrate Rev1 by translesion polymerases ζ and κ.

Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a ß-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.

The unusual UBZ domain of Saccharomyces cerevisiae polymerase η.

Recent research has revealed the presence of ubiquitin-binding domains in the Y family polymerases. The ubiquitin-binding zinc finger (UBZ) domain of human polymerase η is vital for its regulation, localization, and function. Here, we elucidate structural and functional features of the non-canonical UBZ motif of Saccharomyces cerevisiae pol η. Characterization of pol η mutants confirms the importance of the UBZ motif and implies that its function is independent of zinc binding. Intriguingly, we demonstrate that zinc does bind to and affect the structure of the purified UBZ domain, but is not required for its ubiquitin-binding activity. Our finding that this unusual zinc finger is able to interact with ubiquitin even in its apo form adds support to the model that ubiquitin binding is the primary and functionally important activity of the UBZ domain in S. cerevisiae polymerase η. Putative ubiquitin-binding domains, primarily UBZs, are identified in the majority of known pol η homologs. We discuss the implications of our observations for zinc finger structure and pol η regulation.

Unconventional ubiquitin recognition by the ubiquitin-binding motif within the Y family DNA polymerases iota and Rev1.

Translesion synthesis is an essential cell survival strategy to promote replication after DNA damage. The accumulation of Y family polymerases (pol) iota and Rev1 at the stalled replication machinery is mediated by the ubiquitin-binding motifs (UBMs) of the polymerases and enhanced by PCNA monoubiquitination. We report the solution structures of the C-terminal UBM of human pol iota and its complex with ubiquitin. Distinct from other ubiquitin-binding domains, the UBM binds to the hydrophobic surface of ubiquitin centered at L8. Accordingly, mutation of L8A, but not I44A, of ubiquitin abolishes UBM binding. Human pol iota contains two functional UBMs, both contributing to replication foci formation. In contrast, only the second UBM of Saccharomyces cerevisiae Rev1 binds to ubiquitin and is essential for Rev1-dependent cell survival and mutagenesis. Point mutations disrupting the UBM-ubiquitin interaction also impair the accumulation of pol iota in replication foci and Rev1-mediated DNA damage tolerance in vivo.

Eukaryotic translesion polymerases and their roles and regulation in DNA damage tolerance.

DNA repair and DNA damage tolerance machineries are crucial to overcome the vast array of DNA damage that a cell encounters during its lifetime. In this review, we summarize the current state of knowledge about the eukaryotic DNA damage tolerance pathway translesion synthesis (TLS), a process in which specialized DNA polymerases replicate across from DNA lesions. TLS aids in resistance to DNA damage, presumably by restarting stalled replication forks or filling in gaps that remain in the genome due to the presence of DNA lesions. One consequence of this process is the potential risk of introducing mutations. Given the role of these translesion polymerases in mutagenesis, we discuss the significant regulatory mechanisms that control the five known eukaryotic translesion polymerases: Rev1, Pol zeta, Pol kappa, Pol eta, and Pol iota.

Novel conserved motifs in Rev1 C-terminus are required for mutagenic DNA damage tolerance.

The genes encoding Rev1 and DNA polymerase zeta (Rev3/Rev7) are together required for the vast majority of DNA damage-induced mutations in eukaryotes from yeast to humans. Here, we provide insight into the critical role that the Saccharomyces cerevisiae Rev1 C-terminus plays in the process of mutagenic DNA damage tolerance. The Rev1 C-terminus was previously thought to be poorly conserved and therefore not likely to be important for mediating protein-protein interactions. However, through comprehensive alignments of the Rev1 C-terminus, we have identified novel and hitherto unrecognized conserved motifs that we show play an essential role in REV1-dependent survival and mutagenesis in S. cerevisiae, likely in its post-replicative gap-filling mode. We further show that the minimal C-terminal fragment of Rev1 containing these highly conserved motifs is sufficient to interact with Rev7.

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

Eukaryotes 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 Saccharomyces cerevisiae or Schizosaccharomyces 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.

Novel role for the C terminus of Saccharomyces cerevisiae Rev1 in mediating protein-protein interactions.

The Saccharomyces cerevisiae REV3/7-encoded polymerase zeta and Rev1 are central to the replicative bypass of DNA lesions, a process called translesion synthesis (TLS). While yeast polymerase zeta extends from distorted DNA structures, Rev1 predominantly incorporates C residues from across a template G and a variety of DNA lesions. Intriguingly, Rev1 catalytic activity does not appear to be required for TLS. Instead, yeast Rev1 is thought to participate in TLS by facilitating protein-protein interactions via an N-terminal BRCT motif. In addition, higher eukaryotic homologs of Rev1 possess a C terminus that interacts with other TLS polymerases. Due to a lack of sequence similarity, the yeast Rev1 C-terminal region, located after the polymerase domain, had initially been thought not to play a role in TLS. Here, we report that elevated levels of the yeast Rev1 C terminus confer a strong dominant-negative effect on viability and induced mutagenesis after DNA damage, highlighting the crucial role that the C terminus plays in DNA damage tolerance. We show that this phenotype requires REV7 and, using immunoprecipitations from crude extracts, demonstrate that, in addition to the polymerase-associated domain, the extreme Rev1 C terminus and the BRCT region of Rev1 mediate interactions with Rev7.

Increased expression of Ifi202, an IFN-activatable gene, in B6.Nba2 lupus susceptible mice inhibits p53-mediated apoptosis.

Increased expression of p202 protein (encoded by the Ifi202 gene) in splenocytes derived from B6.Nba2 mice (congenic for the Nba2 interval derived from the New Zealand Black mice) was correlated with defects in apoptosis of splenic B cells and increased susceptibility to develop systemic lupus erythematosus. We have now investigated the molecular mechanisms by which increased expression of p202 in B6.Nba2 cells contributes to defects in apoptosis. In this study, we report that increased expression of p202 in the B6.Nba2 splenocytes, as compared with cells derived from the parental C57BL/6 (B6) mice, was correlated with increased levels of p53 protein and inhibition of p53-mediated transcription of target genes that encode proapoptotic proteins. Conversely, knockdown of p202 expression in B6.Nba2 cells resulted in stimulation of p53-mediated transcription. We found that p202 bound to p53 in the N-terminal region (aa 44-83) comprising the proline-rich region that is important for p53-mediated apoptosis. Consistent with the binding of p202 to p53, increased expression of p202 in B6.Nba2 mouse embryonic fibroblasts inhibited UV-induced apoptosis. Taken together, our observations support the idea that increased expression of p202 in B6.Nba2 mice increases the susceptibility to develop lupus, in part, by inhibiting p53-mediated apoptosis.