title: Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions.
abstract: MutY homologue (MYH) is a DNA glycosylase which excises adenine paired with the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG, or G(o)) during base excision repair (BER). Base excision by MYH results in an apurinic/apyrimidinic (AP) site in the DNA where the DNA sugar-phosphate backbone remains intact. A key feature of MYH activity is its physical interaction and coordination with AP endonuclease I (APE1), which subsequently nicks DNA 5' to the AP site. Because AP sites are mutagenic and cytotoxic, they must be processed by APE1 immediately after the action of MYH glycosylase. Our recent reports show that the interdomain connector (IDC) of human MYH (hMYH) maintains interactions with hAPE1 and the human checkpoint clamp Rad9-Rad1-Hus1 (9-1-1) complex. In this study, we used NMR chemical shift perturbation experiments to determine hMYH-binding site on hAPE1. Chemical shift perturbations indicate that the hMYH IDC peptide binds to the DNA-binding site of hAPE1 and an additional site which is distal to the APE1 DNA-binding interface. In these two binding sites, N212 and Q137 of hAPE1 are key mediators of the MYH/APE1 interaction. Intriguingly, despite the fact that hHus1 and hAPE1 both interact with the MYH IDC, hHus1 does not compete with hAPE1 for binding to hMYH. Rather, hHus1 stabilizes the hMYH/hAPE1 complex both in vitro and in cells. This is consistent with a common theme in BER, namely that the assembly of protein-DNA complexes enhances repair by efficiently coordinating multiple enzymatic steps while simultaneously minimizing the release of harmful repair intermediates.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Paz J
authors.initials: PJ
authors.lastname: Luncsford
authors.forename: Brittney A
authors.initials: BA
authors.lastname: Manvilla
authors.forename: Dimeka N
authors.initials: DN
authors.lastname: Patterson
authors.forename: Shuja S
authors.initials: SS
authors.lastname: Malik
authors.forename: Jin
authors.initials: J
authors.lastname: Jin
authors.forename: Bor-Jang
authors.initials: BJ
authors.lastname: Hwang
authors.forename: Randall
authors.initials: R
authors.lastname: Gunther
authors.forename: Snigdha
authors.initials: S
authors.lastname: Kalvakolanu
authors.forename: Leonora J
authors.initials: LJ
authors.lastname: Lipinski
authors.forename: Weirong
authors.initials: W
authors.lastname: Yuan
authors.forename: Wuyuan
authors.initials: W
authors.lastname: Lu
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Drohat
authors.forename: A-Lien
authors.initials: AL
authors.lastname: Lu
authors.forename: Eric A
authors.initials: EA
authors.lastname: Toth
mesh_terms: D001665:Binding Sites; D055162:Biocatalysis; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008279:Magnetic Resonance Imaging; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D055550:Protein Stability
chemical_list: D018797:Cell Cycle Proteins; D004247:DNA; D045647:DNA Glycosylases; C061738:mutY adenine glycosylase; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
keywords: 7,8-dihydro-8-oxoguanine; 8-oxoG or G(o); 9–1–1; AP; AP endonuclease; AP-endonuclease 1; APE1; BER; BSA; Bacillus stearothermophilus MutY; Base excision repair; BstMutY; Checkpoint clamp; DNA glycosylase; Ec-cMutY; Escherichia coli MutY catalytic domain; EtBr; F; GST; HSQC; IDC; LP; MAP; MEF; MYH or MUTYH; MYH-associated polyposis; MutY homologue; ODN; Rad9–Rad1–Hus1; Regulatory protein interactions; S. cerevisiae; S. pombe; SN; Saccharomyces cerevisiae; Schizosaccharomyces pombe; apurinic/apyrimidinic; base excision repair; bovine serum albumin; ethidium bromide; glutathione S-transferase; h; heteronuclear spin quantum coherence; human; interdomain connector; long-patch; mouse embryonic fibroblast; oligodeoxynucleotides; single-nucleotide patch; tetrahydrofuran abasic site analog