title: Destabilization of the PCNA trimer mediated by its interaction with the NEIL1 DNA glycosylase.
abstract: The base excision repair (BER) pathway repairs oxidized lesions in the DNA that result from reactive oxygen species generated in cells. If left unrepaired, these damaged DNA bases can disrupt cellular processes such as replication. NEIL1 is one of the 11 human DNA glycosylases that catalyze the first step of the BER pathway, i.e. recognition and excision of DNA lesions. NEIL1 interacts with essential replication proteins such as the ring-shaped homotrimeric proliferating cellular nuclear antigen (PCNA). We isolated a complex formed between NEIL1 and PCNA (±DNA) using size exclusion chromatography (SEC). This interaction was confirmed using native gel electrophoresis and mass spectrometry. Stokes radii measured by SEC hinted that PCNA in complex with NEIL1 (±DNA) was no longer a trimer. Height measurements and images obtained by atomic force microscopy also demonstrated the dissociation of the PCNA homotrimer in the presence of NEIL1 and DNA, while small-angle X-ray scattering analysis confirmed the NEIL1 mediated PCNA trimer dissociation and formation of a 1:1:1 NEIL1-DNA-PCNA(monomer) complex. Furthermore, ab initio shape reconstruction provides insights into the solution structure of this previously unreported complex. Together, these data point to a potential mechanistic switch between replication and BER.
authors.affiliation: Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, AL 36604-1405, USA.
authors.forename: Aishwarya
authors.initials: A
authors.lastname: Prakash
authors.affiliation: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, USA.
authors.forename: Kedar
authors.initials: K
authors.lastname: Moharana
authors.affiliation: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, USA.
authors.forename: Susan S
authors.initials: SS
authors.lastname: Wallace
authors.affiliation: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Drive, Burlington, VT 05405-0068, USA.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Doublié
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D018625:Microscopy, Atomic Force; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D004247:DNA; D045647:DNA Glycosylases; C473634:NEIL1 protein, human