Dynamics of Gapped DNA Recognition by Human Polymerase β

Abstract

Kinetics of human polymerase ␤ binding to gapped DNA substrates having single stranded (ss) DNA gaps with five or two nucleotide residues in the ssDNA gap has been examined, using the fluorescence stopped-flow technique. The mechanism of the recognition does not depend on the length of the ssDNA gap. Formation of the enzyme complex with both DNA substrates occurs by a minimum three-step reaction, with the bimolecular step followed by two isomerization steps. The results indicate that the polymerase initiates the association with gapped DNA substrates through the DNA-binding subsite located on the 8-kDa domain of the enzyme. This first association step is independent of the length of the ssDNA gap and is characterized by similar rate constants for both examined DNA substrates. The subsequent, first-order transition occurs at the rate of ϳ ϳ600 -1200 s ؊1 . This is the major docking step accompanied by favorable free energy changes in which the 31-kDa domain engages in interactions with the DNA. The 5-terminal PO 4 ؊ group downstream from the primer is not a specific recognition element of the gap. However, the phosphate group affects the enzyme orientation in the complex with the DNA, particularly, for the substrate with a longer gap.

Functional Characterization of the Multidomain F Plasmid TraI Relaxase-Helicase

“…The observed dramatic increase in K N was caused by a statistical factor arising from the existence of multiple potential binding sites, instead of the formation of more contacts between TraI and ssDNA oligomers (28,31,32). This statistical factor can be defined in terms of the intrinsic binding constant (K int ) and the total site size (m) (29,30) as shown in Equation 12.…”

“…To understand the intrinsic DNA-binding property of TraI, we have studied the interaction of TraI with unlabeled DNA oligomers using the MCT method as described by Jezewska and Bujalowski (27) (see also "Experimental Procedures"). This method has been successfully applied to the analysis of the interactions of the PriA and RepA helicases with DNA (29,30). In brief, a fluorescently labeled DNA oligomer, or the reference DNA, is titrated with TraI in the absence or presence of an unlabeled DNA oligomer, or the competing DNA, whose binding parameters are to be determined.…”

Spectral Self-Interference Fluorescence Microscopy to Study Conformation of Biomolecules with Nanometer Accuracy

“…For the short patch repair, the phosphodiester bond at 3′ of the abasic site is cleaved by glycosylate, and the 5′ bond is incised by APE1 endonuclease, which then recruits DNA polymerase to fill the gap that is ligated by a DNA-ligase complex. During the process of BER, the conformation and flexibility of DNA changes with the binding of different enzymes, which kinks the DNA to different angles [168][169][170][171][172][173][174][175]. Through careful design of experiments, the SSFM platform can potentially detect DNA BER through direct detection of DNA conformational changes and protein-binding events.…”

“…Although traditional fluorescence microscopy, like other conventional optical microscopy techniques, cannot provide resolution below the diffraction limit, some new fluorescence imaging techniques have broken the limit. Among these techniques, fluorescence (Förster) resonance energy transfer (FRET) allows investigation of biomolecular structure in vivo with a high temporal resolution when combined with stopped flow techniques [26,27] and provides a measurement of the distance between nearby donor and acceptor molecules in a distance range of about 10-100 Å [28]. However, the techniques previously discussed are not high throughput and therefore are not suited to study DNA sequence dependence, one of the most critical parameters influencing protein-DNA interactions.…”

Informatics and Physics Models of Recognitions of DNA Replication and Their Biological Applications

“…It is well known that scientists have investigated recognitions between a DNA polymerase and an initiation site [1][2][3][4][5][6] and of pairing deoxyribonucleotides [6][7][8][9][10][11] for DNA replication for many decades. But, those studies have been performed in a perspective of molecular biology or biochemistry and the recognition distance is limited to lengths of chemical bounds.…”