Mechanism of interaction between human 8-oxoguanine-DNA glycosylase and AP endonuclease.
Human 8-oxoguanine-DNA glycosylase (OGG1) is the main human base excision protein that removes a mutagenic lesion 8-oxoguanine (8-oxoG) from DNA. Since OGG1 has DNA glycosylase and weak abasic site (AP) lyase activities and is characterized by slow product release, turnover of the enzyme acting alone is low. Recently it was shown that human AP endonuclease (APE1) enhances the activity of OGG1. This enhancement was proposed to be passive, resulting from APE1 binding to or cleavage of AP sites after OGG1 dissociation. Here we present evidence that APE1 could actively displace OGG1 from its product, directly increasing the turnover of OGG1. We have observed that APE1 forms an electrophoretically detectable complex with OGG1 cross-linked to DNA by sodium borohydride. Using oligonucleotide substrates with a single 8-oxoG residue located in their 5'-terminal, central or 3'-terminal part, we have demonstrated that OGG1 activity does not increase only for the first of these three substrates, indicating that APE1 interacts with the DNA stretch 5' to the bound OGG1 molecule. In kinetic experiments, APE1 enhanced the product release constant but not the rate constant of base excision by OGG1. Moreover, OGG1 bound to a tetrahydrofuran analog of an abasic site stimulated the activity of APE1 on this substrate. Using a concatemeric DNA substrate, we have shown that APE1 likely displaces OGG1 in a processive mode, with OGG1 remaining on DNA but sliding away in search for a new lesion. Altogether, our data support a model in which APE1 specifically recognizes an OGG1/DNA complex, distorts a stretch of DNA 5' to the OGG1 molecule, and actively displaces the glycosylase from the lesion.
Sidorenko
Victoria S
VS
SB RAS Institute of Chemical Biology and Fundamental Medicine, 8 Lavrentieva Avenue, Novosibirsk 630090, Russia.
Nevinsky
Georgy A
GA
Zharkov
Dmitry O
DO
D001665:Binding Sites; D001894:Borohydrides / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D001894:Borohydrides; D011994:Recombinant Proteins; C025364:sodium borohydride; D004247:DNA; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase