Mechanistic analysis of PCNA poly-ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5

• Published in: The EMBO journal 2009-12, Vol.28 (23), p.3657-3666
• Main Authors: Parker, Joanne L, Ulrich, Helle D
• Format: Article
• Language: English
• Subjects: Deoxyribonucleic acid; DNA; DNA Helicases - chemistry; DNA Helicases - genetics; DNA Helicases - metabolism; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; EMBO13; EMBO31; Enzymes; Molecular biology; PCNA; poly-ubiquitin chains; Polyubiquitin - chemistry; Polyubiquitin - genetics; Polyubiquitin - metabolism; Proliferating Cell Nuclear Antigen - chemistry; Proliferating Cell Nuclear Antigen - genetics; Proliferating Cell Nuclear Antigen - metabolism; Proteins; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Substrate Specificity - genetics; ubiquitin protein ligase; ubiquitin-conjugating enzyme; Ubiquitin-Conjugating Enzymes - chemistry; Ubiquitin-Conjugating Enzymes - genetics; Ubiquitin-Conjugating Enzymes - metabolism; Ubiquitin-Protein Ligases - metabolism
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1038/emboj.2009.303

Poly‐ubiquitylation is a common post‐translational modification that can impart various functions to a target protein. Several distinct mechanisms have been reported for the assembly of poly‐ubiquitin chains, involving either stepwise transfer of ubiquitin monomers or attachment of a preformed poly‐ubiquitin chain and requiring either a single pair of ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3), or alternatively combinations of different E2s and E3s. We have analysed the mechanism of poly‐ubiquitylation of the replication clamp PCNA by two cooperating E2–E3 pairs, Rad6–Rad18 and Ubc13–Mms2–Rad5. We find that the two complexes act sequentially and independently in chain initiation and stepwise elongation, respectively. While loading of PCNA onto DNA is essential for recognition by Rad6–Rad18, chain extension by Ubc13–Mms2–Rad5 is only slightly enhanced by loading. Moreover, in contrast to initiation, chain extension is tolerant to variations in the attachment site of the proximal ubiquitin moiety. Our results provide information about a unique conjugation mechanism that appears to be specialised for a regulatable pattern of dual modification.