title: PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions.
abstract: Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNAS228Imutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3p66and PolD4p12. In contrast p21 largely retains the ability to bind PCNAS228I. This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNAS228Ibinding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNAS228Ifor specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
authors.forename: Rosemary H C
authors.initials: RH
authors.lastname: Wilson
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Biasutto
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK.
authors.forename: Lihao
authors.initials: L
authors.lastname: Wang
authors.affiliation: Target Discovery Institute, University of Oxford, Roosevelt Drive, Oxford OX3 7FZ, UK.
authors.forename: Roman
authors.initials: R
authors.lastname: Fischer
authors.affiliation: University of Exeter Medical School, Barrack Road, Exeter, EX2 5DW, UK.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Baple
authors.affiliation: University of Exeter Medical School, Barrack Road, Exeter, EX2 5DW, UK.
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Crosby
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK; School of Life Sciences, University of Sussex, Falmer, Brighton, BN1 9RH, UK.
authors.forename: Erika J
authors.initials: EJ
authors.lastname: Mancini
authors.affiliation: Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK. Electronic address: catherine.green@well.ox.ac.uk.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Green
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D004260:DNA Repair; D049914:DNA Repair-Deficiency Disorders / Q000378:metabolism*; D004261:DNA Replication; D006801:Humans; D046912:Multiprotein Complexes; D020125:Mutation, Missense; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs / Q000235:genetics*; D054875:Ubiquitination
chemical_list: C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen; C435720:POLD3 protein, human; C521101:POLD4 protein, human; D004258:DNA Polymerase III
keywords: DNA repair; DNA replication; PCNA; PCNA-associated repair disorder (PARD)