Structure of monoubiquitinated PCNA and implications for translesion synthesis and DNA polymerase exchange

• Published in: Nature Structural & Molecular Biology Vol. 17; no. 4; pp. 479 - 484
• Main Authors: Freudenthal, Bret D, Ramaswamy, S, Washington, M Todd, Gakhar, Lokesh
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-04-2010; Nature Publishing Group
• Subjects: 631/337/1427/2354; 631/45/535; Antigens; Biochemistry; Biological Microscopy; Biomedical and Life Sciences; Causes of; Chemical synthesis; Crystallography, X-Ray; Deoxyribonucleic acid; DNA; DNA damage; DNA polymerases; DNA Repair; DNA-Directed DNA Polymerase - metabolism; Genetic aspects; Ion exchange; Lesions; Life Sciences; Membrane Biology; Models, Molecular; Physiological aspects; Polymers; Proliferating Cell Nuclear Antigen - chemistry; Proliferating Cell Nuclear Antigen - metabolism; Protein Conformation; Protein Structure; Ubiquitin; Ubiquitination; United States
• ISSN: 1545-9993; 1545-9985
• DOI: 10.1038/nsmb.1776

The monoubiquitination of processivity factor PCNA at Lys164 in yeast leads to exchange of a replicative DNA polymerase with a translesion one. Now a functional, split version of monoubiquitinated PCNA is developed and its crystal structure shows that the ubiquitin moiety is located at the back face of the PCNA ring and does not cause large conformational changes in PCNA.
DNA synthesis by classical polymerases can be blocked by many lesions. These blocks are overcome by translesion synthesis, whereby the stalled classical, replicative polymerase is replaced by a nonclassical polymerase. In eukaryotes this polymerase exchange requires proliferating cell nuclear antigen (PCNA) monoubiquitination. To better understand the polymerase exchange, we developed a means of producing monoubiquitinated PCNA, by splitting the protein into two self-assembling polypeptides. We determined the X-ray crystal structure of monoubiquitinated PCNA and found that the ubiquitin moieties are located on the back face of PCNA and interact with it through their canonical hydrophobic surface. Moreover, the attachment of ubiquitin does not change PCNA's conformation. We propose that PCNA ubiquitination facilitates nonclassical polymerase recruitment to the back of PCNA by forming a new binding surface for nonclassical polymerases, consistent with a 'tool belt' model of the polymerase exchange.