RNAs undergo phase transitions with lower critical solution temperatures.

Co-phase separation of RNAs and RNA-binding proteins drives the biogenesis of ribonucleoprotein granules. RNAs can also undergo phase transitions in the absence of proteins. However, the physicochemical driving forces of protein-free, RNA-driven phase transitions remain unclear. Here we report that various types of RNA undergo phase separation with system-specific lower critical solution temperatures. This entropically driven phase separation is an intrinsic feature of the phosphate backbone that requires Mg2+ ions and is modulated by RNA bases. RNA-only condensates can additionally undergo enthalpically favourable percolation transitions within dense phases. This is enabled by a combination of Mg2+-dependent bridging interactions between phosphate groups and RNA-specific base stacking and base pairing. Phase separation coupled to percolation can cause dynamic arrest of RNAs within condensates and suppress the catalytic activity of an RNase P ribozyme. Our work highlights the need to incorporate RNA-driven phase transitions into models for ribonucleoprotein granule biogenesis.

Interlocking activities of DNA polymerase ß in the base excision repair pathway.

SignificanceBase excision repair (BER) is one of the major DNA repair pathways used to fix a myriad of cellular DNA lesions. The enzymes involved in BER, including DNA polymerase ß (Polß), have been identified and characterized, but how they act together to efficiently perform BER has not been fully understood. Through gel electrophoresis, mass spectrometry, and kinetic analysis, we discovered that the two enzymatic activities of Polß can be interlocked, rather than functioning independently from each other, when processing DNA intermediates formed in BER. The finding prompted us to hypothesize a modified BER pathway. Through conventional and time-resolved X-ray crystallography, we solved 11 high-resolution crystal structures of cross-linked Polß complexes and proposed a detailed chemical mechanism for Polß's 5'-deoxyribose-5-phosphate lyase activity.

Purification, reconstitution, and mass analysis of archaeal RNase P, a multisubunit ribonucleoprotein enzyme.

The ubiquitous ribonucleoprotein (RNP) form of RNase P catalyzes the Mg2+-dependent cleavage of the 5' leader of precursor-transfer RNAs. The rate and fidelity of the single catalytic RNA subunit in the RNase P RNP is significantly enhanced by association with protein cofactors. While the bacterial RNP exhibits robust activity at near-physiological Mg2+ concentrations with a single essential protein cofactor, archaeal and eukaryotic RNase P are dependent on up to 5 and 10 protein subunits, respectively. Archaeal RNase P-whose proteins share eukaryotic homologs-is an experimentally tractable model for dissecting in a large RNP the roles of multiple proteins that aid an RNA catalyst. We describe protocols to assemble RNase P from Methanococcus maripaludis, a methanogenic archaeon. We present strategies for tag-less purification of four of the five proteins (the tag from the fifth is removed post-purification), an approach that helps reconstitute the RNase P RNP with near-native constituents. We demonstrate the value of native mass spectrometry (MS) in establishing the accurate masses (including native oligomers and modifications) of all six subunits in M. maripaludis RNase P, and the merits of mass photometry (MP) as a complement to native MS for characterizing the oligomeric state of protein complexes. We showcase the value of native MS and MP in revealing time-dependent modifications (e.g., oxidation) and aggregation of protein subunits, thereby providing insights into the decreased function of RNase P assembled with aged preparations of recombinant subunits. Our protocols and cautionary findings are applicable to studies of other cellular RNPs.

The many faces of RNA-based RNase P, an RNA-world relic.

RNase P is an essential enzyme that catalyzes removal of the 5' leader from precursor transfer RNAs. The ribonucleoprotein (RNP) form of RNase P is present in all domains of life and comprises a single catalytic RNA (ribozyme) and a variable number of protein cofactors. Recent cryo-electron microscopy structures of representative archaeal and eukaryotic (nuclear) RNase P holoenzymes bound to tRNA substrate/product provide high-resolution detail on subunit organization, topology, and substrate recognition in these large, multisubunit catalytic RNPs. These structures point to the challenges in understanding how proteins modulate the RNA functional repertoire and how the structure of an ancient RNA-based catalyst was reshaped during evolution by new macromolecular associations that were likely necessitated by functional/regulatory coupling.

Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P.

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5' leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250-500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10-20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

Kinetic investigation of the polymerase and exonuclease activities of human DNA polymerase ε holoenzyme.

In eukaryotic DNA replication, DNA polymerase ε (Polε) is responsible for leading strand synthesis, whereas DNA polymerases α and δ synthesize the lagging strand. The human Polε (hPolε) holoenzyme is comprised of the catalytic p261 subunit and the noncatalytic p59, p17, and p12 small subunits. So far, the contribution of the noncatalytic subunits to hPolε function is not well understood. Using pre-steady-state kinetic methods, we established a minimal kinetic mechanism for DNA polymerization and editing catalyzed by the hPolε holoenzyme. Compared with the 140-kDa N-terminal catalytic fragment of p261 (p261N), which we kinetically characterized in our earlier studies, the presence of the p261 C-terminal domain (p261C) and the three small subunits increased the DNA binding affinity and the base substitution fidelity. Although the small subunits enhanced correct nucleotide incorporation efficiency, there was a wide range of rate constants when incorporating a correct nucleotide over a single-base mismatch. Surprisingly, the 3'→5' exonuclease activity of the hPolε holoenzyme was significantly slower than that of p261N when editing both matched and mismatched DNA substrates. This suggests that the presence of p261C and the three small subunits regulates the 3'→5' exonuclease activity of the hPolε holoenzyme. Together, the 3'→5' exonuclease activity and the variable mismatch extension activity modulate the overall fidelity of the hPolε holoenzyme by up to 3 orders of magnitude. Thus, the presence of p261C and the three noncatalytic subunits optimizes the dual enzymatic activities of the catalytic p261 subunit and makes the hPolε holoenzyme an efficient and faithful replicative DNA polymerase.

Cancers from Novel Pole-Mutant Mouse Models Provide Insights into Polymerase-Mediated Hypermutagenesis and Immune Checkpoint Blockade.

POLE mutations are a major cause of hypermutant cancers, yet questions remain regarding mechanisms of tumorigenesis, genotype-phenotype correlation, and therapeutic considerations. In this study, we establish mouse models harboring cancer-associated POLE mutations P286R and S459F, which cause rapid albeit distinct time to cancer initiation in vivo, independent of their exonuclease activity. Mouse and human correlates enabled novel stratification of POLE mutations into three groups based on clinical phenotype and mutagenicity. Cancers driven by these mutations displayed striking resemblance to the human ultrahypermutation and specific signatures. Furthermore, Pole-driven cancers exhibited a continuous and stochastic mutagenesis mechanism, resulting in intertumoral and intratumoral heterogeneity. Checkpoint blockade did not prevent Pole lymphomas, but rather likely promoted lymphomagenesis as observed in humans. These observations provide insights into the carcinogenesis of POLE-driven tumors and valuable information for genetic counseling, surveillance, and immunotherapy for patients. SIGNIFICANCE: Two mouse models of polymerase exonuclease deficiency shed light on mechanisms of mutation accumulation and considerations for immunotherapy.See related commentary by Wisdom and Kirsch p. 5459.

Alternative Protein Topology-Mediated Evolution of a Catalytic Ribonucleoprotein.

The high-resolution structures of yeast RNase for mitochondrial RNA processing (MRP), a catalytic ribonucleoprotein (RNP), recently reported by Lan et al. and Perederina et al. illustrate how RNA-mediated selection of alternative protein conformations, sampled during stochastic excursions by polymorphic/metamorphic proteins, enabled RNAs and proteins to mutually influence their functional repertoires and shape RNP evolution.

Dynamic basis for dG•dT misincorporation via tautomerization and ionization.

Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (Genol•T/UG•Tenol/Uenol; population less than 0.4%) and one anionic species (G•T-/U-; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10-2 to 10-3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT- dominant at pH 8.4 and above or for some mutagenic nucleotides.

Comprehensive Analysis of Hypermutation in Human Cancer.

We present an extensive assessment of mutation burden through sequencing analysis of >81,000 tumors from pediatric and adult patients, including tumors with hypermutation caused by chemotherapy, carcinogens, or germline alterations. Hypermutation was detected in tumor types not previously associated with high mutation burden. Replication repair deficiency was a major contributing factor. We uncovered new driver mutations in the replication-repair-associated DNA polymerases and a distinct impact of microsatellite instability and replication repair deficiency on the scale of mutation load. Unbiased clustering, based on mutational context, revealed clinically relevant subgroups regardless of the tumors' tissue of origin, highlighting similarities in evolutionary dynamics leading to hypermutation. Mutagens, such as UV light, were implicated in unexpected cancers, including sarcomas and lung tumors. The order of mutational signatures identified previous treatment and germline replication repair deficiency, which improved management of patients and families. These data will inform tumor classification, genetic testing, and clinical trial design.

Replication studies of carboxymethylated DNA lesions in human cells.

Metabolic activation of some N-nitroso compounds (NOCs), an important class of DNA damaging agents, can induce the carboxymethylation of nucleobases in DNA. Very little was previously known about how the carboxymethylated DNA lesions perturb DNA replication in human cells. Here, we investigated the effects of five carboxymethylated DNA lesions, i.e. O6-CMdG, N6-CMdA, N4-CMdC, N3-CMdT and O4-CMdT on the efficiency and fidelity of DNA replication in HEK293T human embryonic kidney cells. We found that, while neither N6-CMdA nor N4-CMdC blocked DNA replication or induced mutations, N3-CMdT, O4-CMdT and O6-CMdG moderately blocked DNA replication and induced substantial frequencies of T→A (81%), T→C (68%) and G→A (6.4%) mutations, respectively. In addition, our results revealed that CRISPR-Cas9-mediated depletion of Pol η resulted in significant drops in bypass efficiencies of N4-CMdC and N3-CMdT. Diminution in bypass efficiencies was also observed for N6-CMdA and O6-CMdG upon depletion of Pol κ, and for O6-CMdG upon removal of Pol ζ. Together, our study provided molecular-level insights into the impacts of the carboxymethylated DNA lesions on DNA replication in human cells, revealed the roles of individual translesion synthesis DNA polymerases in bypassing these lesions, and suggested the contributions of O6-CMdG, N3-CMdT and O4-CMdT to the mutations found in p53 gene of human gastrointestinal cancers.

Structural basis for the D-stereoselectivity of human DNA polymerase ß.

Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase ß (hPolß), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolß. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolß, DNA and L-dCTP or the triphosphate forms of antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) provide little structural evidence to suggest that hPolß follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolß discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. The two NRTIs escape some of the active site selection through the base and sugar modifications but are selected against through the inability of hPolß to complete thumb domain closure.

Pre-steady-state kinetic investigation of bypass of a bulky guanine lesion by human Y-family DNA polymerases.

3-Nitrobenzanthrone (3-NBA), a byproduct of diesel exhaust, is highly present in the environment and poses a significant health risk. Exposure to 3-NBA results in formation of N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA), a bulky DNA lesion that is of particular importance due to its mutagenic and carcinogenic potential. If not repaired or bypassed during genomic replication, dGC8-N-ABA can stall replication forks, leading to senescence and cell death. Here we used pre-steady-state kinetic methods to determine which of the four human Y-family DNA polymerases (hPolη, hPolκ, hPolι, or hRev1) are able to catalyze translesion synthesis of dGC8-N-ABAin vitro. Our studies demonstrated that hPolη and hPolκ most efficiently bypassed a site-specifically placed dGC8-N-ABA lesion, making them good candidates for catalyzing translesion synthesis (TLS) of this bulky lesion in vivo. Consistently, our publication (Biochemistry 53, 5323-31) in 2014 has shown that small interfering RNA-mediated knockdown of hPolη and hPolκ in HEK293T cells significantly reduces the efficiency of TLS of dGC8-N-ABA. In contrast, hPolι and hRev1 were severely stalled by dGC8-N-ABA and their potential role in vivo was discussed. Subsequently, we determined the kinetic parameters for correct and incorrect nucleotide incorporation catalyzed by hPolη at various positions upstream, opposite, and downstream from dGC8-N-ABA. Notably, nucleotide incorporation efficiency and fidelity both decreased significantly during dGC8-N-ABA bypass and the subsequent extension step, leading to polymerase pausing and error-prone DNA synthesis by hPolη. Furthermore, hPolη displayed nucleotide concentration-dependent biphasic kinetics at the two polymerase pause sites, suggesting that multiple enzyme•DNA complexes likely exist during nucleotide incorporation.

DNA polymerase-mediated synthesis of unbiased threose nucleic acid (TNA) polymers requires 7-deazaguanine to suppress G:G mispairing during TNA transcription.

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9°N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA. However, our ability to transcribe four-nucleotide libraries has been limited by chain termination events that prevent the synthesis of full-length TNA products. Here, we show that chain termination is caused by tG:dG mispairing in the enzyme active site. We demonstrate that the unnatural base analogue 7-deazaguanine (7dG) will suppress tGTP misincorporation by inhibiting the formation of Hoogsteen tG:dG base pairs. DNA templates that contain 7dG in place of natural dG residues replicate with high efficiency and >99% overall fidelity. Pre-steady-state kinetic measurements indicate that the rate of tCTP incorporation is 5-fold higher opposite 7dG than dG and only slightly lower than dCTP incorporation opposite either 7dG or dG. These results provide a chemical solution to the problem of how to synthesize large, unbiased pools of TNA molecules by polymerase-mediated synthesis.

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ.

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'→5' exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Polɛ heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Polɛ in vitro. However, similar studies of the human Polɛ heterotetramer (hPolɛ) have been limited by the difficulty of obtaining hPolɛ in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPolɛ from insect host cells has allowed for isolation of greater amounts of active hPolɛ, thus enabling a more detailed kinetic comparison between hPolɛ and an active N-terminal fragment of the hPolɛ catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPolɛ. We observe that the small subunits increase DNA binding by hPolɛ relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'→5' exonuclease activity of hPolɛ is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPolɛ and sway hPolɛ toward DNA synthesis rather than proofreading.

Significant contribution of the 3'→5' exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ.

Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3'→5' exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 10(6)-10(8) incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3'→5' exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10(-4)-10(-7)) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3'→5' exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 10(2) to 1.2 × 10(4)-fold. The resulting overall fidelity of hPolϵ (10(-6)-10(-11)) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1-1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation.

Structural basis for the binding and incorporation of nucleotide analogs with L-stereochemistry by human DNA polymerase λ.

Although lamivudine and emtricitabine, two L-deoxycytidine analogs, have been widely used as antiviral drugs for years, a structural basis for D-stereoselectivity against L-dNTPs, enantiomers of natural nucleotides (D-dNTPs), by any DNA polymerase or reverse transcriptase has not been established due to lack of a ternary structure of a polymerase, DNA, and an incoming L-dNTP. Here, we report 2.10-2.25 Å ternary crystal structures of human DNA polymerase λ, DNA, and L-deoxycytidine 5'-triphosphate (L-dCTP), or the triphosphates of lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) with four ternary complexes per asymmetric unit. The structures of these 12 ternary complexes reveal that relative to D-deoxycytidine 5'-triphosphate (D-dCTP) in the canonical ternary structure of Polλ-DNA-D-dCTP, L-dCTP, (-)3TC-TP, and (-)FTC-TP all have their ribose rotated by 180°. Among the four ternary complexes with a specific L-nucleotide, two are similar and show that the L-nucleotide forms three Watson-Crick hydrogen bonds with the templating nucleotide dG and adopts a chair-like triphosphate conformation. In the remaining two similar ternary complexes, the L-nucleotide surprisingly interacts with the side chain of a conserved active site residue R517 through one or two hydrogen bonds, whereas the templating dG is anchored by a hydrogen bond with the side chain of a semiconserved residue Y505. Furthermore, the triphosphate of the L-nucleotide adopts an unprecedented N-shaped conformation. Our mutagenic and kinetic studies further demonstrate that the side chain of R517 is critical for the formation of the abovementioned four complexes along proposed catalytic pathways for L-nucleotide incorporation and provide the structural basis for the D-stereoselectivity of a DNA polymerase.

Kinetic mechanism of DNA polymerization catalyzed by human DNA polymerase ε.

Eukaryotes require highly accurate and processive DNA polymerases to ensure faithful and efficient replication of their genomes. DNA polymerase ε (Polε) has been shown to catalyze leading-strand DNA synthesis during replication in vivo, but little is known about the kinetic mechanism of polymerization catalyzed by this replicative enzyme. To elucidate this mechanism, we have generated a truncated, exonuclease-deficient mutant of the catalytic subunit of human Polε (Polε exo-) and carried out pre-steady-state kinetic analysis of this enzyme. Our results show that Polε exo-, as other DNA polymerases, follows an induced-fit mechanism when catalyzing correct nucleotide incorporation. Polε exo- binds DNA with a Kd(DNA) of 79 nM and dissociates from the E·DNA binary complex with a rate constant of 0.021 s(-1). Although Polε exo- binds a correct incoming nucleotide weakly with a Kd(dTTP) of 31 µM, it catalyzes correct nucleotide incorporation at a fast rate constant of 248 s(-1) at 20 °C. Both a large reaction amplitude difference (42%) between pulse-chase and pulse-quench assays and a small elemental effect (0.9) for correct dTTP incorporation suggest that a slow conformational change preceding the chemistry step limits the rate of correct nucleotide incorporation. In addition, our kinetic analysis shows that Polε exo- exhibits low processivity during polymerization. To catalyze leading-strand synthesis in vivo, Polε likely interacts with its three smaller subunits and additional replication factors in order to assemble a replication complex and significantly enhance its polymerization processivity.