Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 290, Issue 23, Pages 14338-14349Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M114.621128
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Funding
- Russian Academy of Sciences Molecular AMP
- Cell Biology Program [6.11, 6.12]
- Russian Foundation for Basic Research [14-04-01879, 13-04-00013, 14-04-31174, 15-04-00467]
- Russian Science Foundation [14-14-00063, 14-24-00093]
- Russian Ministry of Education and Science [SS-1205.2014.4, SP-4012.2013.4]
- Russian Science Foundation [14-24-00093, 14-14-00063] Funding Source: Russian Science Foundation
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Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3'-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln(41) and Leu(81) as DNA lesion sensors.
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