Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn, rather than Mg, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn and Mg, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn than with Mg. Notably, the substitution fidelity of PrimPol in the presence of Mn was determined to be in the range of 3.4×10 to 3.8×10, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn and 150-4500 with Mg, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).
The patient reported the symptoms of pain and blurry vision
3-Nitrobenzanthrone (3-NBA) is a byproduct of diesel exhaust and is highly present in industrial and populated areas. Inhalation of 3-NBA results in formation of N-(2′-deoxyguanosin-8-yl)-3aminobenzanthrone (dG C8-N-ABA), a bulky DNA lesion that is of concern due to its mutagenic and carcinogenic potential. If dG C8-N-ABA is not bypassed during genomic replication, the lesion can stall cellular DNA replication machinery, leading to senescence or apoptosis. We have previously used running start assays to demonstrate that human DNA polymerases eta (hPolκ) and kappa (hPolκ) are able to catalyze translesion DNA synthesis (TLS) across a site-specifically placed dG C8-N-ABA in a DNA template. Consistently, gene knockdown of hPolη and hPolκ in HEK293T cells reduces the efficiency of TLS across dG C8-N-ABA by ~25 and ~30%, respectively. Here, we kinetically investigated why hPolκ paused when bypassing and extending from dG C8-N-ABA. Our kinetic data show that correct dCTP incorporation efficiency of hPolκ dropped by 116-fold when opposite dG C8-N-ABA relative to undamaged dG, leading to hPolκ pausing at the lesion site observed in the running start assays. The already low nucleotide incorporation fidelity of hPolκ was further decreased by 10-fold during lesion bypass and thus, incorrect nucleotides, especially dATP, were incorporated opposite dG C8-N-ABA with comparable efficiencies as correct dCTP. With regard to the dG C8-N-ABA bypass product extension step, hPolκ incorporated correct dGTP onto the damaged DNA substrate with a 786-fold lower efficiency than onto the corresponding undamaged DNA substrate, resulting in hPolκ pausing at the site in the running start assays. Furthermore, hPolκ extended the primer-terminal matched base pair dC:dG C8-N-ABA with a 100-1,000 fold lower fidelity than it extended the undamaged dC:dG base pair. Together, our kinetic results strongly indicate that hPolκ was error-prone during TLS of dG C8-N-ABA .
Ebstein's anomaly is a rare form of cyanotic congenital heart disease (CHD) that involves malformation and dysfunction of the tricuspid valve and right ventricle (RV). The severity of the defect impacts clinical presentation, survival, and treatment options. Presentation during the neonatal period with hypoxemia and cyanosis is noted in patients with severe tricuspid valve malformation, a hypoplastic RV, or RV outflow tract obstruction. However, presentation later in infancy is more common when there is a moderate tricuspid valve malformation and no associated RV outflow tract obstruction. Although Ebstein's anomaly is not generally associated with other congenital defects, patients may occasionally require surgery for other comorbid conditions. We describe the perioperative anesthetic management of an adolescent with Ebstein's anomaly for posterior spinal fusion. Previous reports of anesthetic care in this clinical scenario are reviewed, anesthetic considerations discussed, and options for intraoperative monitoring and anesthetic care presented.
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