Steric Constraints Dependent on Nucleobase Pair Orientation Vary in Different DNA Polymerase Active Sites

Abstract

Finding the right fit: Herein, we report on the development of novel steric probes and present initial insights into their interplay with DNA polymerases. Our findings provide experimental evidence for varied enzyme-substrate interactions that might account for the varied selectivity previously observed.

Importance of the C2, N7, and C8 Positions to the Mutagenic Potential of 8-Oxo-2′-deoxyguanosine with Two A Family Polymerases

“…Thus, the same negative interaction with a large C8 atom that leads to the reduced stability of these base pairs may also help reduce the efficiency of incorporation of dCTP opposite the individual nucleotides. These new findings are also in accordance with much previous work that suggests the high fidelity displayed by A family polymerases stems from them having “tight” insertion sites that are sensitive to steric changes. − While the standard Watson–Crick (W–C) base pairs, dG:dC, dC:dG, dA:dT, and dT:dA, all have similar shapes and H8­(purine)–H5­(pyrimidine) distances, the overall base pair shape and H8–H5 distance would be significantly increased if dCTP is incorporated opposite CldG, BrdG, OdG, SdG, OdI, or SdI. Thus, if the polymerase insertion site is normally rigid and tightly defined around the cognate W–C base pair shape, detrimental clashing interactions could develop as larger atoms are substituted at the C8 position of dG or OdG.…”

Strukturen von DNA‐Polymerasen mit 4′‐alkylierten Nucleotiden im aktiven Zentrum

“…Die Veränderung der Einbaueffizienz durch Einführung einer zusätzlichen Methylengruppe in das natürliche Nucleotid ist deutlich größer als der Unterschied zwischen der Methyl‐ und Ethylmodifikation. Ähnliche Effekte wurden bereits in Studien an Enzymen aus derselben Sequenzfamilie beobachtet 4a,f,g…”

Nucleotides and nucleic acids; oligo- and polynucleotides

“…91 The 5'-triphosphate derivatives of 4'-methyl-and 4'-ethyl-dA and dT have been synthesised and used as steric probes in the active sites of various DNA polymerases, showing that, for example, the more error prone polymerases, such as Dpo4, incorporate the larger triphosphates more readily than selective polymerases, such as Klenow fragment. 92 4'-C-hydroxymethyl-dT has been introduced into gapmer LNA oligonucleotides to assess the effect of RNAse H activity. It was shown that multiple modifications could be made into an LNA oligomer whilst still activating RNAse H recruitment.…”