In response to DNA damage, the Rad6/Rad18
ubiquitin-conjugating complex monoubiquitinates the replication clamp
proliferating cell nuclear antigen (
PCNA) at Lys-164. Although ubiquitination of
PCNA is recognized as an essential step in initiating postreplication repair, the mechanistic relevance of this modification has remained elusive. Here, we describe a robust in vitro system that ubiquitinates yeast
PCNA specifically on Lys-164. Significantly, only those
PCNA clamps that are appropriately loaded around effector
DNA by its loader,
replication factor C, are ubiquitinated. This observation suggests that, in vitro, only
PCNA present at stalled replication forks is ubiquitinated. Ubiquitinated
PCNA displays the same replicative functions as unmodified
PCNA. These functions include loading onto
DNA by
replication factor C, as well as Okazaki fragment synthesis and maturation by the
PCNA-coordinated actions of
DNA polymerase delta, the
flap endonuclease FEN1, and
DNA ligase I. However, whereas the activity of
DNA polymerase zeta remains unaffected by ubiquitination of
PCNA, ubiquitinated
PCNA specifically activates two key
enzymes in translesion synthesis:
DNA polymerase eta, the yeast
Xeroderma pigmentosum ortholog, and Rev1, a deoxycytidyl
transferase that functions in organizing the mutagenic DNA replication machinery. We propose that ubiquitination of
PCNA increases its functionality as a sliding clamp to promote mutagenic DNA replication.