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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 PCNAS228I mutation 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, PolD3 and PolD4. 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 PCNAS228I binding. 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 PCNAS228I for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD.

Original publication

DOI

10.1016/j.dnarep.2016.12.003

Type

Journal article

Journal

DNA Repair

Publisher

Elsevier

Addresses

RHC Wilson, Wellcome Trust Centre for Human Genetics (WT), Nuffield Department of Medicine, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom, AJ Biasutto, Wellcome Trust Centre for Human Genetics (WT), Division of Structural Biology, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom, L Wang, Wellcome Trust Centre for Human Genetics, Nuffield Department of Medicine, Roosevelt Drive, Oxford, OX3 7BN, UK, R Fischer, University of Oxford, Target Discovery Institute, Roosevelt Drive, Oxford, OX3 7FZ, United Kingdom, EL Baple, University of Exeter, Medical School, Barrack Road, Exeter, EX2 5DW, United Kingdom, AH Crosby, University of Exeter, Medical School, Barrack Road, Exeter, EX2 5DW, UK, EJ Mancini, Wellcome Trust Centre for Human Genetics (WT), Division of Structural Biology, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom, EJ Mancini, University of Sussex, School of Life Sciences, Brighton, BN1 9RH, United Kingdom, CM Green, Wellcome Trust Centre for Human Genetics (WT), Nuffield Department of Medicine, Roosevelt Drive, Oxford, OX3 7BN, United Kingdom

Keywords

PCNA, PCNA-associated repair disorder, DNA repair, DNA replication