Use of ATP, dATP and their α-thio derivatives to study DNA ligase adenylation

Abstract

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

Mammalian DNA Ligases

“…Detection of the enzyme-adenylate complex as a radioactively labelled in termediate by SDS-polyacrylamide gel electrophoresis and autoradiography provides a convenient method for the assay of DNA ligases in partially purified protein fractions. ATP-dependent DNA ligases can employ an alogues such as dA TP, but the anomalous complexes formed with some ligases may function poorly in subsequent steps of the DNA-joining reaction (7). The activated AMP residue of the DNA ligase-adenylate intermediate is transferred to the 5' phosphate terminus of a single strand break in double stranded DNA to generate a covalent DNA-AMP complex with a 5' -5' phosphoanhydride bond.…”

Uracil-DNA Glycosylase of Thermoplasma acidophilumDirects Long-Patch Base Excision Repair, Which Is Promoted by Deoxynucleoside Triphosphates and ATP/ADP, into Short-Patch Repair

“…Adenylation efficiency in pmol of nucleotide bonded to enzyme (see Fig. 8A) was calculated by using the extent of adenylation of T4 DNA ligase as an internal standard (43). Reactions were quenched by the addition of SDS to 1% (wt/vol).…”

The α-thio and/or β-γ-hypophosphate analogs of ATP as cofactors of T4 DNA ligase

“…in the ribo-and 2 0 -deoxyribo-series) the first step of the enzymatic reaction, i.e. enzyme adenylation, is quite efficient, however, the relative efficiency of ligase reaction decreases twofold in the presence of ATPaS instead of ATP [15], though the stereochemistry-dependence for the thio-analog of ATP was not established. Based on these results, we have hypothesized that T4 DNA ligase may be a stereoselective enzyme and may exhibit a different activity dependent on P-configuration of the ATPaS diastereoisomer.…”

“…The series of different ATP analogs have been previously tested as substrates, cofactors or inhibitors of variety of enzymes [11,12] including T4 DNA ligase [13][14][15]. Considering its enantioselective properties, T4 DNA ligase was tested with L-ATP and results suggest that the enzyme is adenylated by L-ATP but this enantiomer is not an effective cofactor in the end-joining reaction compared to natural D-ATP [13].…”

“…With respect to this class of compound, athio-analogs of ATP seem to be especially interesting. ATPaS has been tested previously in the context of its activity as a cofactor for T4 DNA ligase [15], however not in the form of pure diastereoisomer. In the present work, not only the importance of the a-phosphorus configuration in a-thio-ATP, but also b,c-bond were estimated.…”