Ubiquitylation of yeast proliferating cell nuclear antigen and its implications for translesion DNA synthesis

Abstract

The Rad6 -Rad18 ubiquitin-conjugating enzyme complex promotes replication through DNA lesions by means of at least three different pathways: the DNA polymerase (Pol) -and -dependent translesion DNA synthesis (TLS) and a Rad5-Mms2-Ubc13-dependent pathway. In DNA-damaged yeast cells proliferating cell nuclear antigen (PCNA) becomes monoubiquitylated at the K164 residue, and genetic studies in yeast have indicated a requirement for this modification in TLS mediated by Pol and Pol . To be able to decipher the role of PCNA monoubiquitylation in the TLS process, we have reconstituted this PCNA modification in vitro from purified yeast proteins. We show that, in addition to the requirement for Rad6 -Rad18, the reaction depends on the loading of the PCNA homotrimeric ring onto the DNA by replication factor C and that all three PCNA monomers become efficiently ubiquitylated. The availability of PCNA monoubiquitylated on all of its three monomers has enabled us to examine the effects of this PCNA modification on DNA synthesis by Pols ␦, , , and Rev1. Contrary to the prevailing ideas that presume a role for PCNA ubiquitylation in the disruption of Pol␦'s binding to PCNA or in the enhancement of the binding affinity of the TLS Pols for PCNA, we find that PCNA ubiquitylation does not affect any of these processes. These observations lead us to suggest a role for PCNA monoubiquitylation in disrupting the PCNA binding of a protein(s) that otherwise is inhibitory to the binding of PCNA by TLS Pols.proliferating cell nuclear antigen ubiquitylation ͉ Rad6 -Rad18 ubiquitin-conjugating enzyme ͉ Rev1 ͉ DNA polymerase ͉ DNA polymerase D NA lesions in the template strand block the progression of replication fork. Genetic and biochemical studies in the yeast Saccharomyces cerevisiae have indicated a preeminent role of Rad6-Rad18 ubiquitin (Ub)-conjugating enzyme complex (1, 2) in promoting replication through DNA lesions (3). Rad6-Rad18-mediated Ub conjugation is indispensable for lesion bypass, which occurs via at least three independent pathways: DNA polymerase (Pol) -and -mediated translesion DNA synthesis (TLS) and a Rad5-Mms2-Ubc13-dependent pathway whose mechanism of action is not known (4).The RAD30 gene of yeast encodes Pol , which is exceptional among eukaryotic TLS Pols in its proficient and relatively error-free ability to replicate through UV-induced cyclobutane pyrimidine dimers (5-8). Consequently, inactivation of Pol in yeast and humans confers enhanced UV mutagenesis (9-13) and in humans results in the cancer-prone syndrome, the variant form of xeroderma pigmentosum (14, 15). Pol , comprising the Rev3 catalytic and Rev7 accessory subunits (16), promotes TLS by extending from the nucleotide inserted opposite DNA lesions by another DNA Pol (17, 18). Rev1, which, like Pol , is a member of the Y family of Pols, differs from the other Pols of this family in its high degree of specificity for inserting a C opposite template G (19, 20). Although Rev1 is strongly inhibited from inserting nucleotides opposite lesions that form a...

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.…”

Eukaryotic DNA damage tolerance and translesion synthesis through covalent modifications of PCNA

“…One study shows that unmodified PCNA is sufficient to stimulate DNA synthesis by Polκ, primarily by reducing the Km to enhance correct nucleotide incorporation [127]. The direct challenge came from the in vitro reconstitution of the DNA synthesis reaction, in which PCNA monoubiquitinated on all three monomers does not enhance affinity for any polymerases examined, nor does it enhance TLS activity by Y-family polymerases [128]. Furthermore, a recent report [129] showed that mutations in the UBZ motif of yeast Polη did not impair its in vivo or in vitro TLS functions.…”

Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation

“…Accumulating lines of evidence suggest that ubiquitylation plays a role in the regulation of PCNA functions. [24][25][26][27][28][29] Ubiquitylation has indeed emerged as an important regulatory switch during DNA replication, and the DNA-damage response specifically involves postreplication DNA repair acting as an error-prone replication safeguard mechanism. 46 Alternatively, mono-ubiquitylation may prevent binding of replication factors to PCNA.…”

“…23 Additionally, PCNA mono and poly-ubiquitylation in response to DNA damage and sumoylation in the absence of damage was described. [24][25][26][27][28][29] Other than forming a homotrimeric structure, PCNA also has been found to form double homotrimeric complexes. [30][31][32] As each monomer has specific protein binding sites, the polymeric structure allows PCNA to bind different partners suggesting that the aggregation status may be relevant for its functions.…”