Bifunctional degraders of cyclin dependent kinase 9 (CDK9): Probing the relationship between linker length, properties, and selective protein degradation.

Cyclin-dependent kinase 9 (CDK9) is a promising therapeutic target in multiple cancer types, including acute myeloid leukemia (AML). Protein degraders, also known as proteolysis targeting chimeras (PROTACs), have emerged as tools for the selective degradation of cancer targets, including CDK9, complementing the activity of traditional small-molecule inhibitors. These compounds typically incorporate previously reported inhibitors and a known E3 ligase ligand to induce ubiquitination and subsequent degradation of the target protein. Although many protein degraders have been reported in the literature, the properties of the linker necessary for efficient degradation still require special attention. In this study, a series of protein degraders was developed, employing the clinically tested CDK inhibitor AT7519. The purpose of this study was to examine the effect that linker composition, specifically chain length, would have on potency. In addition to establishing a baseline of activity for various linker compositions, two distinct homologous series, a fully alkyl series and an amide-containing series, were prepared, demonstrating the dependence of degrader potency in these series on linker length and the correlation with predicted physicochemical properties.

The Falcon Test: An Observer Agreement Study in Subjects With and Without Anterior Knee Pain.

Introduction: A shortened rectus femoris muscle has been associated with many different musculoskeletal problems. Assessing rectus femoris muscle length is commonly performed using the Modified Thomas Test. However, this test position is often difficult to assume and there are difficulties with reliably measuring rectus femoris length. A method that that uses an easier position to assume and could be more reliable would be beneficial to therapists. The purpose of this study was to determine observer agreement using a new test for assessment of rectus femoris length. A second purpose was to determine if those with anterior knee pain have different rectus femoris muscle length than those without anterior knee pain. Method: Fifty-three participants with and without anterior knee pain were enrolled. Rectus femoris muscle length was measured lying prone with the leg measured on the table while the non-measured leg was off the table in a position of 90° hip flexion. Rectus femoris muscle was lengthened by passively bending the knee until a firm end-feel. The angle of knee flexion was then measured. The process was then repeated after a brief rest period. Results: Observer agreement assessing rectus femoris length using this method showed "almost perfect" reliability for both intra- and inter-rater testing: intra-rater: ICC = .99, [CI95: .98-.99], inter-rater: ICC = .96, [CI95: .92- .98]. Agreement for the sub-sample of those with anterior knee pain (N=16) showed "almost perfect" reliability for intra-rater (ICC 1,1 = .98); [CI95: 0.94-.99] and inter-rater reliability (ICC 2,1 = 0.88); [CI95: 0.70 -.95]. No differences were noted in rectus femoris length between those without and those with anterior knee pain (t= 0.82, p> 0.01); [CI95: -7.8 -3.33]; (SEM = 1.3°; MDC=3.6°). Conclusion: This new method of assessing rectus femoris length is reliable between and within raters. No differences were noted in rectus femoris length between those with anterior knee pain and those without.

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.

Optimization of proteomics sample preparation for forensic analysis of skin samples.

We present an efficient protein extraction and in-solution enzymatic digestion protocol optimized for mass spectrometry-based proteomics studies of human skin samples. Human skin cells are a proteinaceous matrix that can enable forensic identification of individuals. We performed a systematic optimization of proteomic sample preparation for a protein-based human forensic identification application. Digestion parameters, including incubation duration, temperature, and the type and concentration of surfactant, were systematically varied to maximize digestion completeness. Through replicate digestions, parameter optimization was performed to maximize repeatability and increase the number of identified peptides and proteins. Final digestion conditions were selected based on the parameters that yielded the greatest percent of peptides with zero missed tryptic cleavages, which benefit the analysis of genetically variable peptides (GVPs). We evaluated the final digestion conditions for identification of GVPs by applying MS-based proteomics on a mixed-donor sample. The results were searched against a human proteome database appended with a database of GVPs constructed from known non-synonymous single nucleotide polymorphisms (SNPs) that occur at known population frequencies. The aim of this study was to demonstrate the potential of our proteomics sample preparation for future implementation of GVP analysis by forensic laboratories to facilitate human identification. SIGNIFICANCE: Genetically variable peptides (GVPs) can provide forensic evidence that is complementary to traditional DNA profiling and be potentially used for human identification. An efficient protein extraction and reproducible digestion method of skin proteins is a key contributor for downstream analysis of GVPs and further development of this technology in forensic application. In this study, we optimized the enzymatic digestion conditions, such as incubation time and temperature, for skin samples. Our study is among the first attempts towards optimization of proteomics sample preparation for protein-based skin identification in forensic applications such as touch samples. Our digestion method employs RapiGest (an acid-labile surfactant), trypsin enzymatic digestion, and an incubation time of 16 h at 37 °C.

Fractionation of DNA and protein from individual latent fingerprints for forensic analysis.

Human touch samples represent a significant portion of forensic DNA casework. Yet, the generally low abundance of genetic material combined with the predominantly extracellular nature of DNA in these samples makes DNA-based forensic analysis exceptionally challenging. Human proteins present in these same touch samples offer an abundant and environmentally-robust alternative. Proteogenomic methods, using protein sequence variants arising from nonsynonymous DNA mutations, have recently been applied to forensic analysis and may represent a viable option looking forward. However, DNA analysis remains the gold standard and any proteomics-based methods would need to consider how DNA could be co-extracted from samples without significant loss. Herein, we describe a simple workflow for the collection, enrichment and fractionation of DNA and protein in latent fingerprint samples. This approach ensures that DNA collected from a latent fingerprint can be analyzed by traditional DNA casework methods, while protein can be proteolytically digested and analyzed via standard liquid chromatography-tandem mass spectrometry-based proteomics methods from the same touch sample. Sample collection from non-porous surfaces (i.e., glass) is performed through the application of an anionic surfactant over the fingermark. The sample is then split into separate DNA and protein fractions following centrifugation to enrich the protein fraction by pelleting skin cells. The results indicate that this workflow permits analysis of DNA within the sample, yet highlights the challenge posed by the trace nature of DNA in touch samples and the potential for DNA to degrade over time. Protein deposited in touch samples does not appear to share this limitation, with robust protein quantities collected across multiple human donors. The quantity and quality of protein remains robust regardless of fingerprint age. The proteomic content of these samples is consistent across individual donors and fingerprint age, supporting the future application of genetically variable peptide (GVP) analysis of touch samples for forensic identification.

An algorithm for random match probability calculation from peptide sequences.

For the past three decades, forensic genetic investigations have focused on elucidating DNA signatures. While DNA has a number of desirable properties (e.g., presence in most biological materials, an amenable chemistry for analysis and well-developed statistics), DNA also has limitations. DNA may be in low quantity in some tissues, such as hair, and in some tissues it may degrade more readily than its protein counterparts. Recent research efforts have shown the feasibility of performing protein-based human identification in cases in which recovery of DNA is challenged; however, the methods involved in assessing the rarity of a given protein profile have not been addressed adequately. In this paper an algorithm is proposed that describes the computation of a random match probability (RMP) resulting from a genetically variable peptide signature. The approach described herein explicitly models proteomic error and genetic linkage, makes no assumptions as to allelic drop-out, and maps the observed proteomic alleles to their expected protein products from DNA which, in turn, permits standard corrections for population structure and finite database sizes. To assess the feasibility of this approach, RMPs were estimated from peptide profiles of skin samples from 25 individuals of European ancestry. 126 common peptide alleles were used in this approach, yielding a mean RMP of approximately 10-2.

A New Normal.

A badly injured young man and his family take an important step toward acceptance.

Comparative analysis of microbiota along the length of the gastrointestinal tract of two tree squirrel species (Sciurus aberti and S. niger) living in sympatry.

Microbiota inhabiting the gastrointestinal (GI) tract of animals has important impacts on many host physiological processes. Although host diet is a major factor influencing the composition of the gut micro-organismal community, few comparative studies have considered how differences in diet influence community composition across the length of the GI tract. We used 16S sequencing to compare the microbiota along the length of the GI tract in Abert's (Sciurus aberti) and fox squirrels (S. niger) living in the same habitat. While fox squirrels are generalist omnivores, the diet of Abert's squirrels is unusually high in plant fiber, particularly in winter when they extensively consume fiber-rich inner bark of ponderosa pine (Pinus ponderosa). Consistent with previous studies, microbiota of the upper GI tract of both species consisted primarily of facultative anaerobes and was less diverse than that of the lower GI tract, which included mainly obligate anaerobes. While we found relatively little differentiation between the species in the microbiota of the upper GI tract, the community composition of the lower GI tract was clearly delineated. Notably, the Abert's squirrel lower GI community was more stable in composition and enriched for microbes that play a role in the degradation of plant fiber. In contrast, overall microbial diversity was higher in fox squirrels. We hypothesize that these disparities reflect differences in diet quality and diet breadth between the species.

Temporal Dynamics of the Soil Metabolome and Microbiome During Simulated Anaerobic Soil Disinfestation.

Significant interest exists in engineering the soil microbiome to attain suppression of soil-borne plant diseases. Anaerobic soil disinfestation (ASD) has potential as a biologically regulated disease control method; however, the role of specific metabolites and microbial community dynamics contributing to ASD mediated disease control is mostly uncharacterized. Understanding the trajectory of co-evolutionary processes leading to syntrophic generation of functional metabolites during ASD is a necessary prelude to the predictive utilization of this disease management approach. Consequently, metabolic and microbial community profiling were used to generate highly dimensional datasets and network analysis to identify sequential transformations through aerobic, facultatively anaerobic, and anaerobic soil phases of the ASD process and distinct groups of metabolites and microorganisms linked with those stages. Transient alterations in abundance of specific microbial groups, not consistently accounted for in previous studies of the ASD process, were documented in this time-course study. Such events initially were associated with increases and subsequent diminution in highly labile metabolites conferred by the carbon input. Proliferation and dynamic compositional changes in the Firmicutes community continued throughout the anaerobic phase and was linked to temporal changes in metabolite abundance including accumulation of small chain organic acids, methyl sulfide compounds, hydrocarbons, and p-cresol with antimicrobial properties. Novel potential modes of disease control during ASD were identified and the importance of the amendment and "community metabolism" for temporally supplying specific classes of labile compounds were revealed.

Time may heal wounds: Aging and life regrets.

Psychological research on regret has focused mostly on the negative emotions associated with troubling past decisions. Because aging is associated with a preference for positive information in attention and memory, investigation into positive emotions elicited by regrets may provide insights into adult developmental changes in subjective experience. The present study investigated age differences in regret-related affect in a survey of adults (n = 629) aged 18-92 years. Positive and negative affect emerged as discrete dimensions of regret-related affect with age trajectories that benefit well-being. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

The Safety of Bypass to Percutaneous Coronary Intervention Facility by Basic Life Support Providers in Patients with ST-Elevation Myocardial Infarction in Prehospital Setting.

BACKGROUND: Most patients transferred from a non-percutaneous coronary intervention (PCI) facility for primary PCI do not meet target reperfusion times. Direct transportation of patients with ST-elevation myocardial infarction (STEMI) from the scene by advanced life support (ALS) paramedics has been shown to improve reperfusion times and outcomes. OBJECTIVE: The aim of this study was to determine whether it is safe to bypass the closest hospital and transport by basic life support (BLS) provider to a PCI facility. METHODS: This was a health records review of consecutive patients transported to a regional PCI center under an STEMI bypass protocol. Under the PCI bypass protocol, patients were eligible if they presented with symptoms of chest pain, a 12-lead electrocardiogram meeting STEMI criteria, and if transported to the regional PCI center within 60 min. The occurrence of predefined adverse events during transport was determined, which included bradycardia < 50 beats/min, tachycardia > 140 beats/min, hypotension, cardiac arrest, and death. RESULTS: There were 46 cases of STEMI bypass between February 2005 and February 2013. Mean transport time was 29.9 min (range 20-62 min). Mean contact-to-balloon time was 95.2 min (range 68-159 min). Twenty-five adverse events occurred in 20 patients during transport. In 16 of the 20 patients, the adverse events were transiently abnormal vital sign requiring no intervention. In 3 of the patients, the adverse event was clinically significant and it is believed that the patient would have benefitted from advanced cardiac life support care not within the scope of practice of the BLS providers. CONCLUSIONS: In our region, STEMI patients can be diagnosed accurately and transported safely on bypass to a PCI center for primary PCI while respecting target reperfusion times.

Kinetic Mechanism of DNA Polymerases: Contributions of Conformational Dynamics and a Third Divalent Metal Ion.

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.

Barriers to Self-Reporting Patient Safety Incidents by Paramedics: A Mixed Methods Study.

BACKGROUND: A minimal amount of research exists examining the extent to which patient safety events occur within paramedicine and even fewer studies investigating patient safety systems for self-reporting by paramedics. The purpose of this study was to identify barriers to paramedic self-reporting of patient safety incidents (PSIs). METHODS: We randomly distributed paper-based surveys among 1,153 paramedics in an Ontario region in Canada. The survey described one of 5 different PSI clinical scenarios (near miss, adverse event, and minor, major or critical patient care variances) and listed 18 potential barriers to self-reporting PSIs as statements presented for rating on a 5-point Likert scale (very significant = 1 - very insignificant = 5). We invited comments on PSI self-reporting with 2 open-ended questions. We analyzed data with descriptive statistics, chi-square tests and Kruskal-Wallis H test. We used an inductive approach to qualitatively analyze emerging themes. RESULTS: We received responses from 1,133 paramedics (98.3%). Almost one third (28.4%) were Advanced Care Paramedics and 45.1% had >10 years' experience. The top 5 barriers to PSI self-reporting (very significant or significant, %) were the fear of being: punished (81.4%), suspended (79.6%), terminated (79.1%), investigated by Ministry of Health and Long-Term Care (78.4%), and decertified (78.0%). Overall, 64.1% responded they would self-report a given PSI. Intention to self-report a PSI varied according to scenario (22.8% near miss, 46.6% adverse event, 74.4% minor, 92.6% major, 95.6% critical). No association was found between level of training (p = 0.55) or years of experience (p = 0.10) and intention to self-report a PSI. Seven themes to improve PSI self-reporting by paramedics emerged from the qualitative data. CONCLUSIONS: A high proportion of fear-based barriers to self-reporting of PSIs exist among this study population. This suggests that a culture change is needed to facilitate the identification of future patient safety threats.

Time-Dependent Extension from an 8-Oxoguanine Lesion by Human DNA Polymerase Beta.

The oxidative DNA lesion 7,8-dihydro-2'-deoxyguanine (8-oxoG) often occurs in double-stranded DNA and poses a threat to genomic integrity due to the ability of 8-oxoG to form stable Watson-Crick base pairs with deoxycytidine (8-oxoG:dC) and Hoogsteen base pairs with deoxyadenosine (8-oxoG:dA). In humans, short-patch base excision repair of 8-oxoG:dA base pairs requires human DNA polymerase ß (hPolß) to bypass 8-oxoG. Previously, we have shown hPolß-catalyzed 8-oxoG bypass to exhibit low fidelity and identified a unique stacking interaction between the newly incorporated nucleotide (dCMP or dAMP) and the templating 8-oxoG. The effect of this stacking on the ability of hPolß to extend from 8-oxoG during long-patch base excision repair was unknown. Here we report pre-steady-state kinetics and time-dependent crystal structures to demonstrate that extension from both 8-oxoG:dC and 8-oxoG:dA base pairs is 18- to 580-fold less efficient compared to 8-oxoG bypass and that extension from 8-oxoG:dC over 8-oxoG:dA is favored by 15-fold. The overall decrease in efficiency of extension relative to 8-oxoG bypass is due to an alternative nucleotide binding conformation in the precatalytic ternary structures (hPolß·DNA·dNTP) for both extension contexts, wherein the incoming nucleotide is bound in either the canonical Watson-Crick base pair or a nonplanar base pair. In addition, the decreased stability of the ternary complex of 8-oxoG:dA extension results in further loss of efficiency when compared to 8-oxoG:dC extension. Therefore, we hypothesize that the inefficient extension from 8-oxoG:dA serves as a newly discovered fidelity checkpoint during base excision repair.

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.

Advances in Structural and Single-Molecule Methods for Investigating DNA Lesion Bypass and Repair Polymerases.

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 ß. 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 Förster 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.

Structural Insights into the Post-Chemistry Steps of Nucleotide Incorporation Catalyzed by a DNA Polymerase.

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 (PPi) 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 ß (hPolß) and provide a structural basis for PPi release. These post-catalytic structures reveal hPolß in an open conformation with PPi bound in the active site, thereby strongly suggesting that the reverse conformational changes occur prior to PPi 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 hPolß in the open conformation, following incorporation of (-)3TC-MP, with a second (-)3TC-TP molecule bound to the active site in the absence of PPi, suggests that nucleotide binding stimulates PPi 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.

Information and Communication Technology Use Is Related to Higher Well-Being Among the Oldest-Old.

OBJECTIVES: Older adults often prioritize socially meaningful goals over informational goals. Thus, we predicted that using information and communication technology (ICT) in service of socially meaningful versus informational goals relates to higher well-being among the oldest-old. METHOD: We surveyed 445 adults aged 80+ (mean = 84, range = 80-93; 64% female; 26% non-White) online or via telephone. Participants reported motivations for ICT use (connect with others, learn new information) and rated their psychological and physical well-being (i.e., life satisfaction, loneliness, goal attainment, subjective health, functional limitations). We conducted regression and mediation analyses to test our hypothesis. RESULTS: Participants used ICT more to connect with friends/family (M = 3.66, SD = 1.28) than to learn information (M = 2.61, SD = 1.44), p < .001. Overall, ICT use predicted higher well-being across outcomes, ps ≤ .008. Motivations for use differentially mediated these effects: Social motivations mediated the relationships between ICT use and psychological well-being, whereas informational motivations mediated the relationships between ICT use and physical well-being. DISCUSSION: Older adults aged 80+ use ICT less than other generations, but may have much to gain. Using social versus informational technologies may enhance multiple aspects of well-being in different ways during very late life. Highlighting such benefits may increase ICT adoption among the oldest-old.

Rat and poultry feeding studies with soybean meal produced from imidazolinone-tolerant (CV127) soybeans.

The safety and nutritional properties of CV127 soybeans were evaluated in rat and broiler feeding studies. Some episodic differences were observed between rats fed CV127, Conquista, and the standard diet for the endpoints examined. None of these differences were considered treatment related, adverse, or biologically meaningful. In general, birds fed diets containing CV127, Conquista, or Monsoy 8001 showed no significant differences in growth and performance response variables. Chickens fed diets containing Coodetec 217 had lower body weight and weight gain for all developmental periods compared to CV127, but no significant differences were found in feed conversion for the two diets during any development period. The results of both feeding studies demonstrate that CV127 soybeans are as safe, wholesome, and nutritionally valuable as the other soybean meals tested, including those varieties for which histories of safe use have been established and well documented.

Viewing Human DNA Polymerase ß Faithfully and Unfaithfully Bypass an Oxidative Lesion by Time-Dependent Crystallography.

One common oxidative DNA lesion, 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), is highly mutagenic in vivo due to its anti-conformation forming a Watson-Crick base pair with correct deoxycytidine 5'-triphosphate (dCTP) and its syn-conformation forming a Hoogsteen base pair with incorrect deoxyadenosine 5'-triphosphate (dATP). Here, we utilized time-resolved X-ray crystallography to follow 8-oxoG bypass by human DNA polymerase ß (hPolß). In the 12 solved structures, both Watson-Crick (anti-8-oxoG:anti-dCTP) and Hoogsteen (syn-8-oxoG:anti-dATP) base pairing were clearly visible and were maintained throughout the chemical reaction. Additionally, a third Mg(2+) appeared during the process of phosphodiester bond formation and was located between the reacting α- and ß-phosphates of the dNTP, suggesting its role in stabilizing reaction intermediates. After phosphodiester bond formation, hPolß reopened its conformation, pyrophosphate was released, and the newly incorporated primer 3'-terminal nucleotide stacked, rather than base paired, with 8-oxoG. These structures provide the first real-time pictures, to our knowledge, of how a polymerase correctly and incorrectly bypasses a DNA lesion.