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Rev1–Polζ-dependent translesion synthesis (TLS) of DNA is crucial for maintaining genome integrity. To elucidate the mechanism by which the two polymerases cooperate in TLS, we determined the cryogenic electron microscopic structure of the Saccharomyces cerevisiaeRev1–Polζ holocomplex in the act of DNA synthesis (3.53 Å). We discovered that a composite N-helix-BRCT module in Rev1 is the keystone of Rev1–Polζ cooperativity, interacting directly with the DNA template–primer and with the Rev3 catalytic subunit of Polζ. The module is positioned akin to the polymerase-associated domain in Y-family TLS polymerases and is set ideally to interact with PCNA. We delineate the full extent of interactions that the carboxy-terminal domain of Rev1 makes with Polζ and identify potential new druggable sites to suppress chemoresistance from first-line chemotherapeutics. Collectively, our results provide fundamental new insights into the mechanism of cooperativity between Rev1 and Polζ in TLS.

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the SaccharomycescerevisiaePolζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31‚ Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein–protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ’s role in TLS and provide a framework for new cancer therapeutics.

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiaePolδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe−4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.

Missense mutations in Leucine-Rich Repeat Kinase 2(LRRK2) cause the majority of familial and some sporadic forms of Parkinson’s disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small-molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive—some with unwanted side effects and unclear clinical outcome—alternative types of LRRK2 inhibitors are lacking. Herein we identify 5′-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons as well as in transgenic C. elegansand D. melanogasterexpressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the future.

Intramembrane-cleaving proteases (I-CLiPs) activate pools of single-pass helical membrane protein signaling precursors that are key in the physiology of prokaryotic and eukaryotic cells. Proteases typically cleave peptide bonds within extended or flexible regions of their substrates, and thus the mechanism underlying the ability of I-CLiPs to hydrolyze the presumably α-helical transmembrane domain (TMD) of these membrane proteins is unclear. Using deep-ultraviolet resonance Raman spectroscopy in combination with isotopic labeling, we show that although predominantly in canonical α-helical conformation, the TMD of the established I-CLiP substrate Gurken displays 310-helical geometry. As measured by microscale thermophoresis, this substrate binds with high affinity to the I-CLiPs GlpG rhomboid and MCMJR1 presenilin homolog in detergent micelles. Binding results in deep-ultraviolet resonance Raman spectra, indicating conformational changes consistent with unwinding of the 310-helical region of the substrate’s TMD. This 310-helical conformation is key for intramembrane proteolysis, as the substitution of a single proline residue in the TMD of Gurken by alanine suppresses 310-helical content in favor of α-helical geometry and abolishes cleavage without affecting binding to the I-CLiP. Complemented by molecular dynamics simulations of the TMD of Gurken, our vibrational spectroscopy data provide biophysical evidence in support of a model in which the transmembrane region of cleavable I-CLiP substrates displays local deviations in canonical α-helical conformation characterized by chain flexibility, and binding to the enzyme results in conformational changes that facilitate local unwinding of the transmembrane helix for cleavage.

DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a heterodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to form a stable four-subunit enzyme (Polζ-d) upon the incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3D architecture and assembly of Polζ and Polζ-d, we employed electron microscopy. We show here how the catalytic and accessory subunits of Polζ and Polζ-d are organized relative to each other. In particular, we show that Polζ-d has a bilobal architecture resembling the replicative polymerases and that Pol32 lies in proximity to Rev7. Collectively, our study provides views of Polζ and Polζ-d and a structural framework for understanding their roles in DNA damage bypass.

Crohn’s disease (CD)–associated variants in the LRRK2gene for risk (N2081D) and for protection (N551K) mediate shared effects in CD and Parkinson’s disease.