PELDOR analysis of enzyme-induced structural changes in damaged DNA duplexes

Abstract

PELDOR (pulsed electron-electron double resonance) spectroscopy was applied to determine spin-spin distances in spin-labeled DNA duplexes (13-mer and 17-mer) containing the damaged sites 8-oxoguanine or uncleavable abasic site analogue tetrahydrofuran. The lesions were located in one strand of the DNA, and two nitroxyl spin labels were attached at the 5'- and 3'-ends of the complementary strand. PELDOR data allow us to obtain distances between the two spin labels in DNAs, which turned out to be around 5 nm for the 13-mer DNA and around 6 nm for 17-mer DNA. Results of PELDOR measurements were supported by molecular dynamics calculations. Study of the interaction of DNA fragments with DNA repair enzyme 8-oxoguanine-DNA glycosylase from E. coli (Fpg protein) showed that this interaction leads to a noticeable decrease of the distance between spin labels, which indicates the enzyme-induced bending of the DNA duplex. This bending may be important for the mechanisms of recognition of damaged sites by DNA repair enzymes.

Substrate specificity of human apurinic/apyrimidinic endonuclease APE1 in the nucleotide incision repair pathway

“…One can see that the positions of the main maximum of all DNA duplexes in the free state are close to 5.0 nm (Table 2 ) and are in line with the previously obtained data for undamaged DNA ( 49 ). A comparison of distances in the APE1–DNA complexes revealed that under these experimental conditions, DNA duplexes partially remain in a free state with a distance of ∼5.0 nm, along with emergence of a substantial amount of conformations with smaller distances.…”

“…Conventional EPR spectra and original PELDOR time traces of the analysed systems were similar to those presented previously ( 36 , 49 ). The PELDOR traces contain a contribution from inter-molecular magnetic dipole–dipole interactions between spin labels of different DNA molecules and a contribution from intra-molecular interactions between two spin labels in the same DNA molecule.…”

“…As demonstrated in another study ( 49 ), spin labels attached to the phosphate groups of 5′ or 3′ end of the duplexes via a linker that has three (P–O5′, O5′–C5′, C5′–C4′) or two (P–O3′, O3′–C3′) bonds is capable of internal rotation. These flexible linkers provide a lot of freedom to the spin-labelled fragment, relative to the DNA duplex.…”

“…The inter-molecular contribution was removed from the experimental time traces in the way it was done in refs. ( 36 , 49 ), and the refined intramolecular contribution to the PELDOR signal, V N ( T ) was thus obtained (Figure 2 ).…”

Lesion Recognition and Cleavage of Damage-Containing Quadruplexes and Bulged Structures by DNA Glycosylases

“…It has been previously shown by pre–steady-state analyses that the recognition of a damaged nucleotide in a duplex substrate is accompanied by a conformational adjustment of the damaged DNA; this process optimizes specific contacts in the enzyme–substrate complex in the case of either Fpg ( Kuznetsov et al, 2007b , 2011 , 2012 ) or OGG1 ( Kuznetsov et al, 2007a , 2014 ; Kuznetsova et al, 2014 ; Lukina et al, 2017 ). The results of the kinetic analysis clearly indicated that Fpg and OGG1 can control substrate specificity via a multi-stage mechanism of recognition of a specific site, accompanied by conformational changes both in the DNA substrate and in the enzyme.…”