Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1

Abstract

In response to DNA damage, the Rad6͞Rad18 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 ␦, the flap endonuclease FEN1, and DNA ligase I. However, whereas the activity of DNA polymerase remains unaffected by ubiquitination of PCNA, ubiquitinated PCNA specifically activates two key enzymes in translesion synthesis: DNA polymerase , 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.DNA replication ͉ postreplication repair ͉ translesion synthesis ͉ ubiquitination ͉ yeast M odification of the replication clamp proliferating cell nuclear antigen (PCNA) has recently emerged as an important regulatory switch during DNA replication and DNA damage response. The critical site of modification on PCNA is Lys-164, and either sumoylation or ubiquitination at this residue can occur (1, 2). Although sumoylation at Lys-164 is proposed to be associated with normal replicative functions, monoubiquitination of this residue by the Rad6͞Rad18 ubiquitin E2͞E3 complex channels DNA damage into the postreplication DNA repair (PRR) pathway (1,3,4). The PRR pathway is comprised of two error-prone branches involving translesion synthesis (TLS) by error-prone DNA polymerases, and an error-free damage avoidance branch that proceeds by fork regression and template switching (5, 6). The latter branch depends on further modification of monoubiquitinated PCNA involving Rad5 and Mms2͞Ubc13 E2͞E3 catalyzed formation of polyubiquitin chains via an unusual Lys-63 ubiquitin linkage (1).Replicative DNA polymerases of the B-family possess a very restrictive active site, which promotes high-fidelity DNA replication but inhibits bypass synthesis of many DNA lesions (5,7,8). On the other hand, Y-family DNA polymerases are particularly adapted to the bypass of DNA lesions because of a more open active site. For instance, DNA polymerase (Pol ), the ortholog of the human Xeroderma pigmentosum protein, can accommodate a cis-syn pyrimidine dimer in its active site allowing facile damage bypass (9). TLS in yeast ...

Regulation of monoubiquitinated PCNA by DUB autocleavage

“…These domains are required by polη and polι (and possibly other Y-family TLS polymerases) for interaction with monoubiquitinated PCNA. Also, in vitro studies have shown that monoubiquitinated PCNA, but not unmodified PCNA, is required for the activation of Rev1 to promote mutagenic DNA replication 13 . Thus, a condition that upregulates PCNA monoubiquitination (such as USP1 knockdown) is likely to increase replication-coupled mutagenesis through the recruitment and activation of multiple TLS polymerases.…”

“…Thus, a model has emerged in which polη binds to monoubiquitinated PCNA and ensures accurate (error-free) replicative bypass of UV lesions. However, other (error-prone) TLS polymerases (such as polι and Rev1) have recently been shown to rely on monoubiquitinated PCNA for their function 12,13 . How cells limit PCNA monoubiquitination and the unwanted deployment of polη and/or other error-prone TLS polymerases in the absence or presence of extrinsic DNA damage during the synthesis of DNA in S phase is not known.…”

Human SHPRH is a ubiquitin ligase for Mms2–Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen

“…To test for this possibility, we first reconstituted the PCNA monoubiquitylation reaction in vitro using purified Rad6-Rad18. As we and others have shown previously for yeast PCNA (26,27), incubation of human PCNA together with Rad6-Rad18, ubiquitin, ubiquitin-activating enzyme, and ATP but with no DNA, did not support the ubiquitylation of PCNA (Fig. 3B, lane 1), suggesting that PCNA had to be loaded onto DNA for PCNA ubiquitylation.…”

Translesion synthesis in mammalian cells

“…However when the template contained an abasic site, ubiquitination of PCNA substantially increased the efficiency of TLS by polη and Rev1 [31].…”

“…Rev1 interacts with polη, ι, κ and Rev7, in all cases via the same domain contained in its C-terminal 150 aa [32][33][34]. It should also be borne in mind that PCNA is a homotrimer, and the available evidence suggests that ubiquitination is an all or nothing process, ie that all three monomers become ubiquitinated in one trimer [25,31]. Each monomer may therefore be able to interact with a different polymerase, providing a "toolbelt" of different polymerases that can attempt to deal with the blocked fork [35] (Figure 1A).…”