Dataset Information

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.

SUBMITTER: Luncsford PJ

PROVIDER: S-EPMC3857741 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions.

Luncsford Paz J PJ Manvilla Brittney A BA Patterson Dimeka N DN Malik Shuja S SS Jin Jin J Hwang Bor-Jang BJ Gunther Randall R Kalvakolanu Snigdha S Lipinski Leonora J LJ Yuan Weirong W Lu Wuyuan W Drohat Alexander C AC Lu A-Lien AL Toth Eric A EA

DNA repair 20131024 12

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 cyt ...[more]

PMID: 24209961

Similar Datasets

Project description:Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication.Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.

Dataset Information

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions.

Publications

Coordination of MYH DNA glycosylase and APE1 endonuclease activities via physical interactions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets