RESUMEN

T:G mismatches in mammals arise primarily from the deamination of methylated CpG sites or the incorporation of improper nucleotides. The process by which repair enzymes such as thymine DNA glycosylase (TDG) identify a canonical DNA base in the incorrect pairing context remains a mystery. However, the abundant contacts of the repair enzymes with the DNA backbone suggest a role for protein-phosphate interaction in the recognition and repair processes, where conformational properties may facilitate the proper interactions. We have previously used 31P NMR to investigate the energetics of DNA backbone BI-BII interconversion and the effect of a mismatch or lesion compared to canonical DNA and found stepwise differences in ΔG of 1-2 kcal/mol greater than equivalent steps in unmodified DNA. We have currently compared our results to substrate dependence for TDG, MBD4, M. HhaI, and CEBPß, testing for correlations to sequence and base-pair dependence. We found strong correlations of our DNA phosphate backbone equilibrium (Keq) to different enzyme kinetics or binding parameters of these varied enzymes, suggesting that the backbone equilibrium may play an important role in mismatch recognition and/or conformational rearrangement and energetics during nucleotide flipping or other aspects of enzyme interrogation of the DNA substrate.

Asunto(s)

Nucleótidos , Timina ADN Glicosilasa , Animales , Conformación Molecular , Nucleótidos/metabolismo , ADN/química , Secuencia de Bases , Timina ADN Glicosilasa/química , Reparación del ADN , Mamíferos/metabolismo

RESUMEN

T:G mismatches in DNA result in humans primarily from deamination of methylated CpG sites. They are repaired by redundant systems, such as thymine DNA glycosylase (TDG) and methyl-binding domain enzyme (MBD4), and maintenance of these sites has been implicated in epigenetic processes. The process by which these enzymes identify a canonical DNA base in the incorrect basepairing context remains a mystery. However, the conserved contacts of the repair enzymes with the DNA backbone suggests a role for protein-phosphate interaction in the recognition and repair processes. We have used 31P NMR to investigate the energetics of DNA backbone BI-BII interconversion, and for this work have focused on alterations to the activation barriers to interconversion and the effect of a mismatch compared with canonical DNA. We have found that alterations to the ΔG of interconversion for T:G basepairs are remarkably similar to U:G basepairs in the form of stepwise differences in ΔG of 1-2 kcal/mol greater than equivalent steps in unmodified DNA, suggesting a universality of this result for TDG substrates. Likewise, we see perturbations to the free energy (∼1 kcal/mol) and enthalpy (2-5 kcal/mol) of activation for the BI-BII interconversion localized to the phosphates flanking the mismatch. Overall our results strongly suggest that the perturbed backbone energetics in T:G basepairs play a significant role in the recognition process of DNA repair enzymes.

Asunto(s)

Timina ADN Glicosilasa , ADN/química , Reparación del ADN , Epigénesis Genética , Humanos , Cinética , Termodinámica , Timina ADN Glicosilasa/química , Timina ADN Glicosilasa/genética , Timina ADN Glicosilasa/metabolismo

RESUMEN

DNA damage has been implicated in numerous human diseases, particularly cancer, and the aging process. Single-base lesions and mismatches in DNA can be cytotoxic or mutagenic and are recognized by a DNA glycosylase during the process of base excision repair. Altered local dynamics and conformational properties in damaged DNAs have previously been suggested to assist in recognition and specificity. Herein, we use solution nuclear magnetic resonance to quantify changes in BI-BII backbone conformational dynamics due to the presence of single-base lesions in DNA, including uracil, dihydrouracil, 1,N6-ethenoadenine, and T:G mismatches. Stepwise changes to the %BII and ΔG of the BI-BII dynamic equilibrium compared to those of unmodified sequences were observed. Additionally, the equilibrium skews toward endothermicity for the phosphates nearest the lesion/mismatched base pair. Finally, the phosphates with the greatest alterations correlate with those most relevant to the repair of enzyme binding. All of these results suggest local conformational rearrangement of the DNA backbone may play a role in lesion recognition by repair enzymes.

Asunto(s)

Disparidad de Par Base , ADN/genética , ADN/metabolismo , Sitios de Unión , ADN/química , ADN Glicosilasas/metabolismo , Reparación del ADN , Humanos , Mutagénesis , Conformación de Ácido Nucleico , Unión Proteica

RESUMEN

OBJECTIVE: To investigate associations between clinical presentations and treatment of tick paralysis in dogs and cats, particularly the relationship between the dose of tick antiserum (TAS) and time to recovery. DESIGN: A retrospective analysis of 325 cases of tick paralysis (227 dogs, 98 cats), from 2001 to 2010, was conducted using records from six practices in Sydney, New South Wales. RESULTS: Doses of TAS in excess of 2 mL/animal, 0.30 mL/kg and 1.25 mL/tick in dogs, and 2 mL/animal, 0.45 mL/kg and 0.38 mL/tick in cats did not significantly alter the time to recovery. In dogs, a longer time to recovery was associated with cases in winter than in other seasons (P = 0.0099) and with more severe gait scores (P = 0.0002). There was a trend of longer recovery times in patients with higher respiratory scores (P = 0.0561). In cats, a longer time to recovery was associated with multiple ticks (P = 0.0133) and more severe gait scores (P < 0.0001). CONCLUSION: Within this retrospective study, minimum doses of TAS were considered optimal, negating any association between dose rate and time to recovery.