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Base excision repair (BER) is the primary pathway by which eukaryotic cells resolve single base damage. One common example of single base damage is 8-oxo-7,8-dihydro-2'-deoxoguanine (8-oxoG). High incidence and mutagenic potential of 8-oxoG necessitate rapid and efficient DNA repair. How BER enzymes coordinate their activities to resolve 8-oxoG damage while limiting cytotoxic BER intermediates from propagating genomic instability remains unclear. Here we use single-molecule Forster resonance energy transfer (smFRET) and ensemble-level techniques to characterize the activities and interactions of consecutive BER enzymes important for repair of 8-oxoG. In addition to characterizing the damage searching and processing mechanisms of human 8-oxoguanine glycosylase 1 (hOGG1), our data support the existence of a ternary complex between hOGG1, the damaged DNA substrate, and human AP endonuclease 1 (APE1). Our results indicate that hOGG1 is actively displaced from its abasic site containing product by protein-protein interactions with APE1 to ensure timely repair of damaged DNA.

Interest in CRISPR/Cas9 remains high level as new applications of the revolutionary gene-editing tool continue to emerge. While key structural and biochemical findings have illuminated major steps in the enzymatic mechanism of Cas9, several important details remain unidentified or poorly characterized that may contribute to known functional limitations. Here we describe the foundation of research that has led to a fundamental understanding of Cas9 and address mechanistic uncertainties that restrict continued development of this gene-editing platform, including specificity for the protospacer adjacent motif, propensity for off-target binding and cleavage, as well as interactions with cellular components during gene editing. Discussion of these topics and considerations should inspire future research to hone this remarkable technology and advance CRISPR/Cas9 to new heights.

The discovery of prokaryotic adaptive immunity prompted widespread use of the RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) endonuclease Cas9 for genetic engineering. However, its kinetic mechanism remains undefined, and details of DNA cleavage are poorly characterized. Here, we establish a kinetic mechanism of Streptococcus pyogenes Cas9 from guide-RNA binding through DNA cleavage and product release. Association of DNA to the binary complex of Cas9 and guide-RNA is rate-limiting during the first catalytic turnover, while DNA cleavage from a pre-formed ternary complex of Cas9, guide-RNA, and DNA is rapid. Moreover, an extremely slow release of DNA products essentially restricts Cas9 to be a single-turnover enzyme. By simultaneously measuring the contributions of the HNH and RuvC nuclease activities of Cas9 to DNA cleavage, we also uncovered the kinetic basis by which HNH conformationally regulates the RuvC cleavage activity. Together, our results provide crucial kinetic and functional details regarding Cas9 which will inform gene-editing experiments, guide future research to understand off-target DNA cleavage by Cas9, and aid in the continued development of Cas9 as a biotechnological tool.

Proliferating cell nuclear antigen (PCNA) is a trimeric ring-shaped clamp protein that encircles DNA and interacts with many proteins involved in DNA replication and repair. Despite extensive structural work to characterize the monomeric, dimeric, and trimeric forms of PCNA alone and in complex with interacting proteins, no structure of PCNA in a ring-open conformation has been published. Here, we use a multidisciplinary approach, including single-molecule Förster resonance energy transfer (smFRET), native ion mobility-mass spectrometry (IM-MS), and structure-based computational modeling, to explore the conformational dynamics of a model PCNA from Sulfolobus solfataricus (Sso), an archaeon. We found that Sso PCNA samples ring-open and ring-closed conformations even in the absence of its clamp loader complex, replication factor C, and transition to the ring-open conformation is modulated by the ionic strength of the solution. The IM-MS results corroborate the smFRET findings suggesting that PCNA dynamics are maintained in the gas phase and further establishing IM-MS as a reliable strategy to investigate macromolecular motions. Our molecular dynamic simulations agree with the experimental data and reveal that ring-open PCNA often adopts an out-of-plane left-hand geometry. Collectively, these results implore future studies to define the roles of PCNA dynamics in DNA loading and other PCNA-mediated interactions.

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 β (hPolβ), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolβ. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolβ, 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 hPolβ follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolβ 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 hPolβ to complete thumb domain closure.

DNA polymerases are essential enzymes that faithfully and efficiently replicate genomic information.(1–3) The mechanism of nucleotide incorporation by DNA polymerases has been extensively studied structurally and kinetically, but several key steps following phosphodiester bond formation remain structurally uncharacterized due to utilization of natural nucleotides. It is thought that the release of pyrophosphate (PP(i)) triggers reverse conformational changes in a polymerase in order to complete a full catalytic cycle as well as prepare for DNA translocation and subsequent incorporation events. Here, by using the triphosphates of chain-terminating antiviral drugs lamivudine ((−)3TC-TP) and emtricitabine ((−)FTC-TP), we structurally reveal the correct sequence of post-chemistry steps during nucleotide incorporation by human DNA polymerase β (hPolβ) and provide a structural basis for PP(i) release. These post-catalytic structures reveal hPolβ in an open conformation with PP(i) bound in the active site, thereby strongly suggesting that the reverse conformational changes occur prior to PP(i) release. The results also help to refine the role of the newly discovered third divalent metal ion for DNA polymerase-catalyzed nucleotide incorporation. Furthermore, a post-chemistry structure of hPolβ in the open conformation, following incorporation of (−)3TC-MP, with a second (−)3TC-TP molecule bound to the active site in the absence of PP(i), suggests that nucleotide binding stimulates PP(i) dissociation and occurs before polymerase translocation. Our structural characterization defines the order of the elusive post-chemistry steps in the canonical mechanism of a DNA polymerase.

Innovative advances in X-ray crystallography and single-molecule biophysics have yielded unprecedented insight into the mechanisms of DNA lesion bypass and damage repair. Time-dependent X-ray crystallography has been successfully applied to view the bypass of 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxoG), a major oxidative DNA lesion, and the incorporation of the triphosphate form, 8-oxo-dGTP, catalyzed by human DNA polymerase β. Significant findings of these studies are highlighted here, and their contributions to the current mechanistic understanding of mutagenic translesion DNA synthesis (TLS) and base excision repair are discussed. In addition, single-molecule Forster resonance energy transfer (smFRET) techniques have recently been adapted to investigate nucleotide binding and incorporation opposite undamaged dG and 8-oxoG by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. The mechanistic response of Dpo4 to a DNA lesion and the complex smFRET technique are described here. In this perspective, we also describe how time-dependent X-ray crystallography and smFRET can be used to achieve the spatial and temporal resolutions necessary to answer some of the mechanistic questions that remain in the fields of TLS and DNA damage repair.

DNA polymerases catalyze DNA synthesis through a stepwise kinetic mechanism that begins with binding to DNA, followed by selection, binding, and incorporation of a nucleotide into an elongating primer. It is hypothesized that subtle active site adjustments in a polymerase to align reactive moieties limit the rate of correct nucleotide incorporation. DNA damage can impede this process for many DNA polymerases, causing replication fork stalling, genetic mutations, and potentially cell death. However, specialized Y-family DNA polymerases are structurally evolved to efficiently bypass DNA damage in vivo, albeit at the expense of replication fidelity. Dpo4, a model Y-family polymerase from Sulfolobus solfataricus, has been well-studied kinetically, structurally, and computationally, which yielded a mechanistic understanding of how the Y- family DNA polymerases achieve their unique catalytic properties. We previously employed a real-time Förster resonance energy transfer (FRET) technique to characterize the global conformational motions of Dpo4 during DNA binding as well as nucleotide binding and incorporation by monitoring changes in distance between sites on the polymerase and DNA, and even between domains of Dpo4. Here, we extend the utility of our FRET methodology to observe conformational transitions within individual domains of Dpo4 during DNA binding and nucleotide incorporation. The results of this novel, intradomain FRET approach unify findings from many studies to fully clarify the complex DNA binding mechanism of Dpo4. Furthermore, intradomain motions in the Finger domain during nucleotide binding and incorporation, for the first time, report on the rate-limiting step of a single-nucleotide addition catalyzed by Dpo4.

Y-family DNA polymerases are known to bypass DNA lesions in vitro and in vivo and rescue stalled DNA replication machinery. Dpo4, a well-characterized model Y-family DNA polymerase, is known to catalyze translesion synthesis across a variety of DNA lesions including 8-oxo-7,8-dihydro-2′-deoxyguanine (8-oxo-dG). Our previous X-ray crystallographic, stopped-flow Förster resonance energy transfer (FRET), and computational simulation studies have revealed that Dpo4 samples a variety of global conformations as it recognizes and binds DNA. Here we employed single-molecule FRET (smFRET) techniques to investigate the kinetics and conformational dynamics of Dpo4 when it encountered 8-oxo-dG, a major oxidative lesion with high mutagenic potential. Our smFRET data indicated that Dpo4 bound the DNA substrate in multiple conformations, as suggested by three observed FRET states. An incoming correct or incorrect nucleotide affected the distribution and stability of these states with the correct nucleotide completely shifting the equilibrium towards a catalytically competent complex. Furthermore, the presence of the 8-oxo-dG lesion in the DNA stabilized both the binary and ternary complexes of Dpo4. Thus, our smFRET analysis provided a basis for the enhanced efficiency which Dpo4 is known to exhibit when replicating across from 8-oxo-dG.

Faithful transmission and maintenance of genetic material is primarily fulfilled by DNA polymerases. During DNA replication, these enzymes catalyze incorporation of deoxynucleotides into a DNA primer strand based on Watson-Crick complementarity to the DNA template strand. Through the years, research on DNA polymerases from every family and reverse transcriptases has revealed structural and functional similarities, including a conserved domain architecture and purported two-metal-ion mechanism for nucleotidyltransfer. However, it is equally clear that DNA polymerases possess distinct differences that often prescribe a particular cellular role. Indeed, a unified kinetic mechanism to explain all aspects of DNA polymerase catalysis, including DNA binding, nucleotide binding and incorporation, and metal-ion-assisted nucleotidyltransfer (i.e., chemistry), has been difficult to define. In particular, the contributions of enzyme conformational dynamics to several mechanistic steps and their implications for replication fidelity are complex. Moreover, recent time-resolved X-ray crystallographic studies of DNA polymerases have uncovered a third divalent metal ion present during DNA synthesis, the function of which is currently unclear and debated within the field. In this review, we survey past and current literature describing the structures and kinetic mechanisms of DNA polymerases from each family to explore every major mechanistic step while emphasizing the impact of enzyme conformational dynamics on DNA synthesis and replication fidelity. This also includes brief insight into the structural and kinetic techniques utilized to study DNA polymerases and RTs. Furthermore, we present the evidence for the two-metal-ion mechanism for DNA polymerase catalysis prior to interpreting the recent structural findings describing a third divalent metal ion. We conclude by discussing the diversity of DNA polymerase mechanisms and suggest future characterization of the third divalent metal ion to dissect its role in DNA polymerase catalysis.