Structural Insights into DNA Polymerase β Deterrents for Misincorporation Support an Induced-Fit Mechanism for Fidelity

AbstractDNA polymerases generally select the correct nucleotide from a pool of structurally similar molecules to preserve Watson-Crick base-pairing rules. We report the structure of DNA polymerase β with DNA mismatches situated in the polymerase active site. This was achieved by using nicked product DNA that traps the mispair (template-primer, A-C or T-C) in the nascent base pair binding pocket. The structure of each mispair complex indicates that the bases do not form hydrogen bonds with one another, but form a staggered arrangement where the bases of the mispair partially overlap. This prevents closure/opening of the N subdomain that is believed to be required for catalytic cycling. The partially open conformation of the N subdomain results in distinct hydrogen bonding networks that are unique for each mispair. These structures define diverse molecular aspects of misinsertion that are consistent with the induced-fit model for substrate specificity

Slow Conformational Dynamics at C2′-endo Nucleotides in RNA

RNA molecules undergo local conformational dynamics on timescales spanning picoseconds to minutes. Slower local motions have the greater potential to govern RNA folding, ligand recognition, and ribonucleoprotein assembly reactions but are difficult to detect in large RNAs with complex structures. RNA SHAPE chemistry employs acylation of the ribose 2'-hydroxyl position to measure local nucleotide flexibility in RNA and is well-characterized by a mechanism in which each nucleotide samples unreactive (closed) and reactive (open) states. We monitor RNA conformational dynamics over distinct time domains by varying the electrophilicity of the acylating reagent. Select C2'-endo nucleotides are nonreactive toward fast reagents but reactive toward slower SHAPE reagents in both model RNAs and in a large RNA with a tertiary fold. We conclude, first, that the C2'-endo conformation by itself does not govern SHAPE reactivity. However, some C2'-endo nucleotides undergo extraordinarily slow conformational changes, on the order of 10(-4) s(-1). Due to their distinctive local dynamics, C2'-endo nucleotides have the potential to function as rate-determining molecular switches and are likely to play central, currently unexplored, roles in RNA folding and function

The Vehicle, Fall 2006

Table of Contents Ferris WheelEmily Daviscover HerStephen Jefferiespage 1 UntitledBob Freyderpage 2 Writing at O\u27BrienWillie Joseph Morrispage 3 Blanks and HabitsRebecca M. Griffithpage 4 Soldier\u27s NightmareCraig A. Dennispage 5 UntitledLindsey Durbinpage 6 A Slow, Painless DeathJacob Fosterpage 7 ThoughtAmanda Yealepage 8 The SociopathBob Freyderpage 9 EasyRebecca M. Griffithpage 10 My PartnerDiedre Mapespage 11 BarriersSuzanne Krahnpage 12 The mind is a prisonJordan Hohespage 13 We Were Shirtless When Thousands DiedMitch Jamespage 14 ComplaintAmanda Yealepage 15 UntitledBob Freyderpage 16 MarkedAmanda Yealepage 17 She Wears Red Lipstick, He, Heartsick EyesRebecca M. Griffithpage 18 PrayerAmanda Yealepage 19 HomeDeej Rolewskipage 20 Your DreamDiedre Mapespage 21 Even Fingers Get LonelySuzanne Krahnpage 22 AggressivityMitch Jamespage 23 FallenMitch Jamespage 24 CollapseMario Podeschipage 36 The Italian CrisisAndy Masterspage 41 About the Authorshttps://thekeep.eiu.edu/vehicle/1084/thumbnail.jp

The Vehicle, Fall 2006

Table of Contents Ferris WheelEmily Daviscover HerStephen Jefferiespage 1 UntitledBob Freyderpage 2 Writing at O\u27BrienWillie Joseph Morrispage 3 Blanks and HabitsRebecca M. Griffithpage 4 Soldier\u27s NightmareCraig A. Dennispage 5 UntitledLindsey Durbinpage 6 A Slow, Painless DeathJacob Fosterpage 7 ThoughtAmanda Yealepage 8 The SociopathBob Freyderpage 9 EasyRebecca M. Griffithpage 10 My PartnerDiedre Mapespage 11 BarriersSuzanne Krahnpage 12 The mind is a prisonJordan Hohespage 13 We Were Shirtless When Thousands DiedMitch Jamespage 14 ComplaintAmanda Yealepage 15 UntitledBob Freyderpage 16 MarkedAmanda Yealepage 17 She Wears Red Lipstick, He, Heartsick EyesRebecca M. Griffithpage 18 PrayerAmanda Yealepage 19 HomeDeej Rolewskipage 20 Your DreamDiedre Mapespage 21 Even Fingers Get LonelySuzanne Krahnpage 22 AggressivityMitch Jamespage 23 FallenMitch Jamespage 24 CollapseMario Podeschipage 36 The Italian CrisisAndy Masterspage 41 About the Authorshttps://thekeep.eiu.edu/vehicle/1084/thumbnail.jp

Template strand scrunching during DNA gap repair synthesis by human polymerase λ

Family X polymerases such as DNA polymerase (Pol ) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5' and 3' ends of short gaps. DNA binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair

Identification and functional characterization of polymorphisms in human cyclooxygenase-1 (PTGS1)

Cyclooxygenase-1 (COX-1, PTGS1) catalyzes the conversion of arachidonic acid to prostaglandin H2, which is subsequently metabolized to various biologically active prostaglandins. We sought to identify and characterize the functional relevance of genetic polymorphisms in PTGS1

The structural basis for partitioning of the XRCC1/DNA ligase III-α BRCT-mediated dimer complexes

The ultimate step common to almost all DNA repair pathways is the ligation of the nicked intermediate to form contiguous double-stranded DNA. In the mammalian nucleotide and base excision repair pathways, the ligation step is carried out by ligase III-α. For efficient ligation, ligase III-α is constitutively bound to the scaffolding protein XRCC1 through interactions between the C-terminal BRCT domains of each protein. Although structural data for the individual domains has been available, no structure of the complex has been determined and several alternative proposals for this interaction have been advanced. Interpretation of the models is complicated by the formation of homodimers that, depending on the model, may either contribute to, or compete with heterodimer formation. We report here the structures of both homodimer complexes as well as the heterodimer complex. Structural characterization of the heterodimer formed from a longer XRCC1 BRCT domain construct, including residues comprising the interdomain linker region, revealed an expanded heterodimer interface with the ligase III-α BRCT domain. This enhanced linker-mediated binding interface plays a significant role in the determination of heterodimer/homodimer selectivity. These data provide fundamental insights into the structural basis of BRCT-mediated dimerization, and resolve questions related to the organization of this important repair complex

Theory of mind in emerging reading comprehension: a longitudinal study of early indirect and direct effects

The relation between children’s theory of mind (ToM) and emerging reading comprehension was investigated in a longitudinal study over 2.5 years. 80 children were tested for ToM, decoding, language skills and executive function (EF) at Time 1 (mean age = 3:10 years). At Time 2 (mean age = 6:03 years) children's word reading efficiency, language skills and reading comprehension were measured. Mediation analysis showed that ToM at Time 1, when children were around four years old, indirectly predicted Time 2 reading comprehension, when children were six years old, via language ability, after controlling for age, non-verbal ability, decoding, EF and earlier language ability. Importantly, ToM at four years old also directly predicted reading comprehension two and a half years later at six years. This is the first longitudinal study to show a direct contribution of theory of mind to reading comprehension in typical development. Findings are discussed in terms of the Simple View of Reading (SVR); ToM not only supports reading comprehension indirectly by facilitating language, but also directly contributes to it over and above the SVR. The potential role of metacognition is considered when accounting for the direct contribution of early ToM to later reading comprehension

An International Multicenter Cohort Study on beta-Blockers for the Treatment of Symptomatic Children With Catecholaminergic Polymorphic Ventricular Tachycardia

Background: Symptomatic children with catecholaminergic polymorphic ventricular tachycardia (CPVT) are at risk for recurrent arrhythmic events. β-Blockers decrease this risk, but studies comparing individual β-blockers in sizeable cohorts are lacking. We aimed to assess the association between risk for arrhythmic events and type of β-blocker in a large cohort of symptomatic children with CPVT.Methods: From 2 international registries of patients with CPVT, RYR2 variant–carrying symptomatic children (defined as syncope or sudden cardiac arrest before β-blocker initiation and age at start of β-blocker therapy &lt;18 years), treated with a β-blocker were included. Cox regression analyses with time-dependent covariates for β-blockers and potential confounders were used to assess the hazard ratio (HR). The primary outcome was the first occurrence of sudden cardiac death, sudden cardiac arrest, appropriate implantable cardioverter-defibrillator shock, or syncope. The secondary outcome was the first occurrence of any of the primary outcomes except syncope.Results: We included 329 patients (median age at diagnosis, 12 [interquartile range, 7–15] years, 35% females). Ninety-nine (30.1%) patients experienced the primary outcome and 74 (22.5%) experienced the secondary outcome during a median follow-up of 6.7 (interquartile range, 2.8–12.5) years. Two-hundred sixteen patients (66.0%) used a nonselective β-blocker (predominantly nadolol [n=140] or propranolol [n=70]) and 111 (33.7%) used a β1-selective β-blocker (predominantly atenolol [n=51], metoprolol [n=33], or bisoprolol [n=19]) as initial β-blocker. Baseline characteristics did not differ. The HRs for both the primary and secondary outcomes were higher for β1-selective compared with nonselective β-blockers (HR, 2.04 [95% CI, 1.31–3.17]; and HR, 1.99 [95% CI, 1.20–3.30], respectively). When assessed separately, the HR for the primary outcome was higher for atenolol (HR, 2.68 [95% CI, 1.44–4.99]), bisoprolol (HR, 3.24 [95% CI, 1.47–7.18]), and metoprolol (HR, 2.18 [95% CI, 1.08–4.40]) compared with nadolol, but did not differ from propranolol. The HR of the secondary outcome was only higher in atenolol compared with nadolol (HR, 2.68 [95% CI, 1.30–5.55]).Conclusions: β1-selective β-blockers were associated with a significantly higher risk for arrhythmic events in symptomatic children with CPVT compared with nonselective β-blockers, specifically nadolol. Nadolol, or propranolol if nadolol is unavailable, should be the preferred β-blocker for treating symptomatic children with CPVT.</p