Mechanistic analysis of PCNA poly-ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5.
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.
Parker
Joanne L
JL
Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms, Herts, UK.
Ulrich
Helle D
HD
D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025821:Polyubiquitin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity / Q000235:genetics; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C063607:RAD18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025821:Polyubiquitin; C471606:RAD6 protein, S cerevisiae; C471610:UBC13 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C076225:RAD5 protein, S cerevisiae; D004265:DNA Helicases; C114683:UFD2 protein, S cerevisiae