The Effect of Socioeconomic Deprivation on Radiographic Deformities in Children With Blount Disease.

BACKGROUND: Blount disease can occur at any time during the growth process, primarily with a bimodal distribution in children younger than 4 years old and adolescents. The disease process most commonly presents in Black adolescents, with disease severity positively correlated with obesity. Given the known associations among race, obesity, and socioeconomic status, we investigated the relationship between the degree of social deprivation and severity of lower extremity deformities among a community-based cohort with Blount disease. METHODS: A retrospective review of hospital records and radiographs of patients with previously untreated Blount disease was conducted. Patients were classified as having early-onset or late-onset Blount disease based on whether the lower limb deformity was noted before or after the age of 4 years. The area deprivation index (ADI), a nationally validated measure that assesses socioeconomic deprivation by residential neighborhood, was calculated for each patient as a surrogate for socioeconomic status. Higher state (range: 1 to 10) or national (range: 1 to 100) ADI corresponds to increased social deprivation. Full-length standing radiographs from index clinic visits were evaluated by 2 reviewers to measure frontal plane deformity. The association of ADI with various demographic and radiographic parameters was then analyzed. RESULTS: Of the 65 patients with Blount disease, 48 (74%) children were Black and 17 (26%) were non-black children. Nineteen children (32 limbs) had early-onset and 46 children (62 limbs) had late-onset disease. Black patients had significantly higher mean state (7.6 vs. 5.4, P =0.009) and national (55.1 vs. 37.4, P =0.002) ADI values than non-black patients. Patients with severe socioeconomic deprivation had significantly greater mechanical axis deviation (66 mm vs. 51 mm, P =0.008). After controlling demographic and socioeconomic factors, the results of multivariate linear regression showed that only increased body mass index (ß=0.19, 95% CI: 0.12-0.26, P <.001) and state ADI (ß=0.021, 95% CI: 0.01-0.53, P =.043) were independently associated with greater varus deformity. CONCLUSIONS: Socioeconomic deprivation was strongly associated with increased severity of varus deformity in children with late-onset Blount disease. Our analysis suggests that obesity and socioeconomic factors are the most influential with regard to disease progression. LEVEL OF EVIDENCE: Level III.

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding.

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

Pain Self-Efficacy (PSEQ) score of <22 is associated with daily opioid use, back pain, disability, and PROMIS scores in patients presenting for spine surgery.

BACKGROUND CONTEXT: Pain self-efficacy, or the belief that one can carry out activities despite pain, has been shown to be associated with back and neck pain severity. However, the literature correlating psychosocial factors to opioid use, barriers to proper opioid use, and Patient-Reported Outcome Measurement Information System (PROMIS) scores is sparse. PURPOSE: The primary aim of this study was to determine whether pain self-efficacy is associated with daily opioid use in patients presenting for spine surgery. The secondary aim was to determine whether there exists a threshold self-efficacy score that is predictive of daily preoperative opioid use and subsequently to correlate this threshold score with opioid beliefs, disability, resilience, patient activation, and PROMIS scores. PATIENT SAMPLE: Five hundred seventy-eight elective spine surgery patients (286 females; mean age of 55 years) from a single institution were included in this study. STUDY DESIGN/SETTING: Retrospective review of prospectively collected data. OUTCOME MEASURES: PROMIS scores, daily opioid use, opioid beliefs, disability, patient activation, resilience. METHODS: Elective spine surgery patients at a single institution completed questionnaires preoperatively. Pain self-efficacy was measured by the Pain Self-Efficacy Questionnaire (PSEQ). Threshold linear regression with Bayesian information criteria was utilized to identify the optimal threshold associated with daily opioid use. Multivariable analysis controlled for age, sex, education, income, and Oswestry Disability Index (ODI) and PROMIS-29, version 2 scores. RESULTS: Of 578 patients, 100 (17.3%) reported daily opioid use. Threshold regression identified a PSEQ cutoff score of <22 as predictive of daily opioid use. On multivariable logistic regression, patients with a PSEQ score <22 had two times greater odds of being daily opioid users than those with a score ≥22. Further, PSEQ <22 was associated with lower patient activation; increased leg and back pain; higher ODI; higher PROMIS pain, fatigue, depression, and sleep scores; and lower PROMIS physical function and social satisfaction scores (p<.05 for all). CONCLUSIONS: In patients presenting for elective spine surgery, a PSEQ score of <22 is associated with twice the odds of reporting daily opioid use. Further, this threshold is associated with greater pain, disability, fatigue, and depression. A PSEQ score <22 can identify patients at high risk for daily opioid use and can guide targeted rehabilitation to optimize postoperative quality of life.

8-Oxo-2'-deoxyguanosine Replication in Mutational Hot Spot Sequences of the p53 Gene in Human Cells Is Less Mutagenic than That of the Corresponding Formamidopyrimidine.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-OxodGuo) is a ubiquitous DNA damage formed by oxidation of 2'-deoxyguanosine. In this study, plasmid DNA containing 8-OxodGuo located in three mutational hot spots of human cancers, codons 248, 249, and 273 of the Tp53 tumor suppressor gene, was replicated in HEK 293T cells. 8-OxodGuo was only a weak block of replication, and the bypass was largely error-free. The mutations (1-5%) were primarily G → T transversions, and the mutation frequency was generally lower than that of the chemically related Fapy·dG. A unique 8-OxodGuo mutation spectrum was observed at each site, as reflected by replication in translesion synthesis (TLS) polymerase- or hPol λ-deficient cells. In codon 248 (CG*G) and 249 (AG*G), where G* denotes 8-OxodGuo, hPol η and hPol ζ carried out largely error-free bypass of the lesion, whereas hPol κ and hPol ι were involved mostly in error-prone TLS, resulting in G → T mutations. 8-OxodGuo bypass in codon 273 (CG*T) was unlike the other two sites, as hPol κ participated in the mostly error-free bypass of the lesion. Yet, in all three sites, including codon 273, simultaneous deficiency of hpol κ and hPol ι resulted in reduction of G → T transversions. This indicates a convincing role of these two TLS polymerases in error-prone bypass of 8-OxodGuo. Although the dominant mutation was G → T in each site, in codon 249, and to a lesser extent in codon 248, significant semi-targeted single-base deletions also occurred, which suggests that 8-OxodGuo can initiate slippage of a base near the lesion site. This study underscores the importance of sequence context in 8-OxodGuo mutagenesis in human cells. It also provides a more comprehensive comparison between 8-OxodGuo and the sister lesion, Fapy·dG. The greater mutagenicity of the latter in the same sequence contexts indicates that Fapy·dG is a biologically significant lesion and biomarker on par with 8-OxodGuo.

Histone Deacetylase 1 Inhibition by Peptides Containing a DNA Damage-Induced, Nonenzymatic, Histone Covalent Modification.

Treatment of HeLa cells with the DNA damaging agent, bleomycin (BLM), results in the formation of a nonenzymatic 5-methylene-2-pyrrolone histone covalent modification on lysine residues (KMP). KMP is much more electrophilic than other N-acyllysine covalent modifications and post-translational modifications, including N-acetyllysine (KAc). Using histone peptides containing KMP, we show that this modification inhibits the class I histone deacetylase, HDAC1, by reacting with a conserved cysteine (C261) located near the active site. HDAC1 is inhibited by histone peptides whose corresponding N-acetylated sequences are known deacetylation substrates, but not one containing a scrambled sequence. The HDAC1 inhibitor, trichostatin A, competes with covalent modification by the KMP-containing peptides. HDAC1 is also covalently modified by a KMP-containing peptide in a complex milieu. These data indicate that peptides containing KMP are recognized by HDAC1 and are bound in the active site. The effects on HDAC1 indicate that KMP formation in cells may contribute to the biological effects of DNA damaging agents, such as BLM, that form this nonenzymatic covalent modification.

A Five-Year Cost-Utility Analysis Comparing Synthetic Cage Versus Allograft Use in Anterior Cervical Discectomy and Fusion Surgery for Cervical Spondylotic Myelopathy.

STUDY DESIGN: Retrospective cost-utility analysis. OBJECTIVE: To conduct a cost-analysis comparing synthetic cage (SC) versus allograft (Allo) over a five-year time horizon. SUMMARY OF BACKGROUND DATA: SC and Allo are two commonly used interbody choices for anterior cervical discectomy and fusion (ACDF) surgery. Previous analyses comparative analyses have reached mixed conclusions regarding their cost-effectiveness, yet recent estimates provide high-quality evidence. MATERIALS AND METHODS: A decision-analysis model comparing the use of Allo versus SC was developed for a hypothetical 60-year-old patient with cervical spondylotic myelopathy undergoing single-level ACDF surgery. A comprehensive literature review was performed to estimate probabilities, costs (2020 USD) and quality-adjusted life years (QALYs) gained over a five-year period. A probabilistic sensitivity analysis using a Monte Carlo simulation of 1000 patients was carried out to calculate incremental cost-effectiveness ratio and net monetary benefits. One-way deterministic sensitivity analysis was performed to estimate the contribution of individual parameters to uncertainty in the model. RESULTS: The use of Allo was favored in 81.6% of the iterations at a societal willing-to-pay threshold of 50,000 USD/QALY. Allo dominated (higher net QALYs and lower net costs) in 67.8% of the iterations. The incremental net monetary benefits in the Allo group was 2650 USD at a willing-to-pay threshold of 50,000 USD/QALY. One-way deterministic sensitivity analysis revealed that the cost of the index surgery was the only factor which significantly contributed to uncertainty. CONCLUSION: Cost-utility analysis suggests that Allo maybe a more cost-effective option compared with SCs in adult patients undergoing ACDF for cervical spondylotic myelopathy.

Covalent Modification of Bromodomain Proteins by Peptides Containing a DNA Damage-Induced, Histone Post-Translational Modification.

An electrophilic 5-methylene-2-pyrrolone modification (KMP ) is produced at lysine residues of histone proteins in nucleosome core particles upon reaction with a commonly formed DNA lesion (C4-AP). The nonenzymatic KMP modification is also generated in the histones of HeLa cells treated with the antitumor agent, bleomycin that oxidizes DNA and forms C4-AP. This nonenzymatic covalent histone modification has the same charge as the N-acetyllysine (KAc ) modification but is more electrophilic. In this study we show that KMP -containing histone peptides are recognized by, and covalently modify bromodomain proteins that are KAc readers. Distinct selectivity preferences for covalent bromodomain modification are observed following incubation with KMP -containing peptides of different sequence. MS/MS analysis of 3 covalently modified bromodomain proteins confirmed that Cys adduction was selective. The modified Cys was not always proximal to the KAc binding site, indicating that KMP -containing peptide interaction with bromodomain protein is distinct from the former. Analysis of protein adduction yields as a function of bromodomain pH at which the protein charge is zero (pI) or cysteine solvent accessible surface area are also consistent with non-promiscuous interaction between the proteins and electrophilic peptides. These data suggest that intracellular formation of KMP could affect cellular function and viability by modifying proteins that regulate genetic expression.

Perplexing Lesion of the Conjunctiva.

An unusual-appearing tumor of the conjunctiva presented in a healthy 11-year-old boy. It was cystic, orange in color, and well encapsulated. After 3 weeks of no response to topical cortisone drops, an excisional biopsy was performed. The histopathology showed the lesion to be a benign lymphoid hyperplasia. [J Pediatr Ophthalmol Strabismus. 2022;59(1):e15-e16.].

Sequence context effects of replication of Fapy•dG in three mutational hot spot sequences of the p53 gene in human cells.

Fapy•dG and 8-OxodGuo are formed in DNA from a common N7-dG radical intermediate by reaction with hydroxyl radical. Although cellular levels of Fapy•dG are often greater, its effects on replication are less well understood than those of 8-OxodGuo. In this study plasmid DNA containing Fapy•dG in three mutational hotspots of human cancers, codons 248, 249, and 273 of the p53 tumor suppressor gene, was replicated in HEK 293T cells. TLS efficiencies for the Fapy•dG containing plasmids varied from 72 to 89%, and were further reduced in polymerase-deficient cells. The mutation frequency (MF) of Fapy•dG ranged from 7.3 to 11.6%, with G→T and G→A as major mutations in codons 248 and 249 compared to primarily G→T in codon 273. Increased MF in hPol ι-, hPol κ-, and hPol ζ-deficient cells suggested that these polymerases more frequently insert the correct nucleotide dC opposite Fapy•dG, whereas decreased G→A in codons 248 and 249 and reduction of all mutations in codon 273 in hPol λ-deficient cells indicated hPol λ's involvement in Fapy•dG mutagenesis. In vitro kinetic analysis using isolated translesion synthesis polymerases and hPol λ incompletely corroborated the mutagenesis experiments, indicating codependence on other proteins in the cellular milieu. In conclusion, Fapy•dG mutagenesis is dependent on the DNA sequence context, but its bypass by the TLS polymerases is largely error-free.

Suppression of DNA Polymerase ß Activity Is Synthetically Lethal in BRCA1-Deficient Cells.

People whose cells express mutated forms of the BRCA1 tumor suppressor are at a higher risk for developing cancer. BRCA1-deficient cells are defective in DNA double-strand break repair. The inhibition of poly(ADP-ribose) polymerase 1 in such cells is a synthetically lethal, cytotoxic effect that has been exploited to produce anticancer drugs such as Olaparib. However, alternative synthetic lethal approaches are necessary. We report that DNA polymerase ß (Pol ß) forms a synthetically lethal interaction with BRCA1. The SiRNA knockdown of Pol ß or the treatment with a Pol ß pro-inhibitor (pro-1) is cytotoxic in BRCA1-deficient ovarian cancer cells. BRCA1-complemented cells are significantly less susceptible to either treatment. pro-1 is also toxic to BRCA1-deficient breast cancer cells, and its toxicity in BRCA1-deficient cells is comparable to that of Olaparib. These experiments establish Pol ß as a synthetically lethal target within BRCA1-deficient cells and a potentially useful one for treating cancer.

Selective Inhibition of DNA Polymerase ß by a Covalent Inhibitor.

DNA polymerase ß (Pol ß) plays a vital role in DNA repair and has been closely linked to cancer. Selective inhibitors of this enzyme are lacking. Inspired by DNA lesions produced by antitumor agents that inactivate Pol ß, we have undertaken the development of covalent small-molecule inhibitors of this enzyme. Using a two-stage process involving chemically synthesized libraries, we identified a potent irreversible inhibitor (14) of Pol ß (KI = 1.8 ± 0.45 µM, kinact = (7.0 ± 1.0) × 10-3 s-1). Inhibitor 14 selectively inactivates Pol ß over other DNA polymerases. LC-MS/MS analysis of trypsin digests of Pol ß treated with 14 identified two lysines within the polymerase binding site that are covalently modified, one of which was previously determined to play a role in DNA binding. Fluorescence anisotropy experiments show that pretreatment of Pol ß with 14 prevents DNA binding. Experiments using a pro-inhibitor (pro-14) in wild type mouse embryonic fibroblasts (MEFs) indicate that the inhibitor (5 µM) is itself not cytotoxic but works synergistically with the DNA alkylating agent, methylmethanesulfonate (MMS), to kill cells. Moreover, experiments in Pol ß null MEFs indicate that pro-14 is selective for the target enzyme. Finally, pro-14 also works synergistically with MMS and bleomycin to kill HeLa cells. The results suggest that pro-14 is a potentially useful tool in studies of the role of Pol ß in disease.

Identifying Poly(ADP-ribose)-Binding Proteins with Photoaffinity-Based Proteomics.

Post-translational modification of proteins with poly(ADP-ribose) (PAR) is an important component of the DNA damage response. Four PAR synthesis inhibitors have recently been approved for the treatment of breast, ovarian, and prostate cancers. Despite the clinical significance of PAR, a molecular understanding of its function, including its binding partners, remains incomplete. In this work, we synthesized a PAR photoaffinity probe that captures and isolates endogenous PAR binders. Our method identified dozens of known PAR-binding proteins and hundreds of novel candidates involved in DNA repair, RNA processing, and metabolism. PAR binding by eight candidates was confirmed using pull-down and/or electrophoretic mobility shift assays. Using PAR probes of defined lengths, we detected proteins that preferentially bind to 40-mer versus 8-mer PAR, indicating that polymer length may regulate the outcome and timing of PAR signaling pathways. This investigation produces the first census of PAR-binding proteins, provides a proteomics analysis of length-selective PAR binding, and associates PAR binding with RNA metabolism and the formation of biomolecular condensates.

Free Falling: Declining Inflation-Adjusted Payment for Arthroplasty Surgeons.

BACKGROUND: Over the past decade, there have been ongoing concerns over declining surgeon compensation for lower extremity arthroplasty. We aimed to determine changes in surgeon payment, patient charges, and overall reimbursement rates for patients undergoing unicompartmental arthroplasty (UKA) and both primary and revision total knee (TKA) and hip (THA) arthroplasty. METHODS: Using Medicare data from 2012 to 2017, we determined inflation-adjusted changes in annual surgeon payment (professional fee), patient charges, and reimbursement rate (payment-to-charge ratio) for UKA and primary/revision TKA and THA. Both nonweighted and weighted (by procedure frequency/volume) means were calculated. RESULTS: Inflation-adjusted surgeon payment decreased for all procedures analyzed, with primary TKA (-17%) and THA (-11%) falling the most. Payment for UKA increased the most (+30%). There was a small increase in charges for THA revision (+2.2%, +2.1%, and +3.2% for acetabulum only, femur only, and both components, respectively). Charges for primary TKA (-3.7%) and THA (-1.5%) decreased slightly. The reimbursement rate for all procedures fell with UKA (-15%), TKA (-14%), and THA (-10%) falling the most. After weighting by procedure frequency/volume and combining all surgeries, average charges fell slightly (-0.7%), whereas surgeon payment (-13%) and reimbursement rate (-12%) fell more sharply. CONCLUSION: Although patient charges have grown in pace with the inflationary rate for primary and revision TKA and THA, surgeon payment and reimbursement rates have fallen sharply. The orthopedic community needs to be aware of these financial trends to communicate to payers and health care policy makers the importance of protecting a sustainable payment infrastructure.

Effect of Histone Lysine Methylation on DNA Lesion Reactivity in Nucleosome Core Particles.

Lysine methylation is a common post-translational histone modification that regulates transcription and gene expression. The lysine residues in the histone tail also react with damaged nucleotides in chromatin, including abasic sites and N7-methyl-2'-deoxyguanosine, the major product of DNA methylating agents. Lysine monomethylation transforms the ε-amine into a secondary amine, which could be more nucleophilic and/or basic than the ε-amine in lysine, and therefore more reactive with damaged DNA. The effect of lysine methylation on the reactivity with abasic sites and N7-methyl-2'-deoxyguanosine was examined in nucleosome core particles using a methylated lysine analogue derived from cysteine. ε-Amine methylation increases the rate constant for abasic site reaction within nucleosome core particles. Reactivity at the two positions examined increased less than twofold. Mechanistic experiments indicate that faster ß-elimination from an intermediate iminium ion accounts for accelerated abasic reactivity. The rate constants for nucleophilic attack (Schiff base/iminium ion formation) by the lysine and methylated lysine analogues are indistinguishable. Similarly, the rate constants describing nucleophilic attack by the lysine and methylated lysine analogues on ß-2'-fluoro-N7-methyl-2'-deoxyguanosine to form DNA-protein cross-links are also within experimental error of one another. These data indicate that abasic site containing DNA will be destabilized by lysine methylation. However, these experiments do not indicate that DNA-protein cross-link formation, a recently discovered form of damage resulting from N7-guanine methylation, will be affected by this post-translational modification.

Ultrasonography of a ureteral and bladder fungal granuloma caused by Scedosporium apiospermum in a basset hound.

A 5-year-old, spayed female, Basset Hound was referred for evaluation of a urinary bladder mass. Ultrasonographic images revealed a large, inhomogeneous, hypoechoic mass associated with the dorsal wall of the neck of the urinary bladder and left ureter. Partial cystectomy and left ureteral reimplantation were performed. Histopathology showed a severe inflammatory mass lesion forming multiple granulomas. A DNA sequencing test revealed Scedosporium apiospermum as the causative agent. Susceptibility tests on the isolated strain indicated susceptibility to voriconazole. To the authors' knowledge, this is the first report describing S. apiospermum as a cause of granulomas involving the canine ureterovesicular junction.

Clitoral cyst in a bitch.

Clitoral cyst can be easily mistaken for a vaginal mass lesion and should be considered in the differential diagnosis for a female dog presenting with an anatomically abnormal external genital examination.

Mechanistic Insight through Irreversible Inhibition: DNA Polymerase θ Uses a Common Active Site for Polymerase and Lyase Activities.

DNA polymerase Î¸ (Pol Î¸) is a multifunctional enzyme. It is nonessential in normal cells, but its upregulation in cancer cells correlates with cellular resistance to oxidative damage and poor prognosis. Pol Î¸ possesses polymerase activity and poorly characterized lyase activity. We examined the Pol Î¸ lyase activity on various abasic sites and determined that the enzyme is inactivated upon attempted removal of the oxidized abasic site commonly associated with C4'-oxidation (pC4-AP). Covalent modification of Pol Î¸ by the DNA lesion enabled determination of the primary nucleophile (Lys2383) responsible for Schiff base formation in the lyase reaction. Unlike some other base excision repair polymerases, Pol Î¸ uses a single active site for polymerase and lyase activity. Mutation of Lys2383 significantly reduces both enzyme activities but not DNA binding. Demonstration that Lys2383 is required for polymerase and lyase activities indicates that this residue is an Achilles heel for Pol Î¸ and suggests a path forward for designing inhibitors of this attractive anticancer target.

An oxidized abasic lesion inhibits base excision repair leading to DNA strand breaks in a trinucleotide repeat tract.

Oxidative DNA damage and base excision repair (BER) play important roles in modulating trinucleotide repeat (TNR) instability that is associated with human neurodegenerative diseases and cancer. We have reported that BER of base lesions can lead to TNR instability. However, it is unknown if modifications of the sugar in an abasic lesion modulate TNR instability. In this study, we characterized the effects of the oxidized sugar, 5'-(2-phosphoryl-1,4-dioxobutane)(DOB) in CAG repeat tracts on the activities of key BER enzymes, as well as on repeat instability. We found that DOB crosslinked with DNA polymerase ß and inhibited its synthesis activity in CAG repeat tracts. Surprisingly, we found that DOB also formed crosslinks with DNA ligase I and inhibited its ligation activity, thereby reducing the efficiency of BER. This subsequently resulted in the accumulation of DNA strand breaks in a CAG repeat tract. Our study provides important new insights into the adverse effects of an oxidized abasic lesion on BER and suggests a potential alternate repair pathway through which an oxidized abasic lesion may modulate TNR instability.

The A-Rule and Deletion Formation During Abasic and Oxidized Abasic Site Bypass by DNA Polymerase θ.

DNA polymerase θ (Pol θ) is implicated in various cellular processes including double-strand break repair and apurinic/apyrimidinic site bypass. Because Pol θ expression correlates with poor cancer prognosis, the ability of Pol θ to bypass the C4'-oxidized abasic site (C4-AP) and 2-deoxyribonolactone (L), which are generated by cytotoxic agents, is of interest. Translesion synthesis and subsequent extension by Pol θ past C4-AP or L and an abasic site (AP) or its tetrahydrofuran analogue (F) was examined. Pol θ conducts translesion synthesis on templates containing AP and F with similar efficiencies and follows the "A-rule," inserting nucleotides in the order A > G > T. Translesion synthesis on templates containing C4-AP and L is less efficient than AP and F, and the preference for A insertion is reduced for L and absent for C4-AP. Extension past all abasic lesions (AP, F, C4-AP, and L) was significantly less efficient than translesion synthesis and yielded deletions caused by the base one or two nucleotides downstream from the lesion being used as a template, with the latter being favored. These results suggest that bypass of abasic lesions by Pol θ is highly mutagenic.