Opposing Effects of Ubiquitin Conjugation and SUMO Modification of PCNA on Replicational Bypass of DNA Lesions in Saccharomyces cerevisiae

Abstract

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases (Pol) and Pol and postreplicational repair mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Here we report our studies with a proliferating cell nuclear antigen (PCNA) mutation, pol30-119, which results from a change of the lysine 164 residue to arginine. It has been shown recently that following treatment of yeast cells with DNA-damaging agents, the lysine 164 residue of PCNA becomes monoubiquitinated in a Rad6-Rad18-dependent manner and that subsequently this PCNA residue is polyubiquitinated via a lysine 63-linked ubiquitin chain in an Mms2-Ubc13-, Rad5-dependent manner. PCNA is also modified by SUMO conjugation at the lysine 164 residue. Our genetic studies with the pol30-119 mutation show that in addition to conferring a defect in Pol-dependent UV mutagenesis and in Pol-dependent TLS, this PCNA mutation inhibits postreplicational repair of discontinuities that form in the newly synthesized strand across from UV lesions. In addition, we provide evidence for the activation of the RAD52 recombinational pathway in the pol30-119 mutant and we infer that SUMO conjugation at the lysine 164 residue of PCNA has a role in suppressing the Rad52-dependent postreplicational repair pathway.Proliferating cell nuclear antigen (PCNA) is the eukaryotic sliding clamp required for processive DNA synthesis. The Saccharomyces cerevisiae POL30 gene encoding PCNA is essential for cell viability (7), and conditional lethal pol30 mutations confer defects in DNA replication (1, 3). PCNA is loaded onto the template-primer junctions of DNA by replication factor C in an ATP-dependent reaction. DNA polymerase ␦ (Pol␦) then binds PCNA and carries out processive DNA synthesis (6, 23). In reconstituted systems containing viral origin sequences, PCNA and Pol␦ carry out replication of both the leading and lagging strands (26,38). In addition to its essential role in DNA replication, PCNA has been shown to be required for various DNA repair processes, including nucleotide excision repair (NER), base excision repair, and mismatch repair (23); more recently, PCNA has been shown to interact physically and functionally with the various translesion synthesis (TLS) polymerases of yeasts and humans (9-11, 13).Genetic studies of S. cerevisiae have indicated that PCNA is involved in RAD6-dependent error-free postreplicative bypass of UV-damaged DNA (35). Postreplicative bypass processes come into play when the DNA replicational machinery encounters a DNA lesion in the template strand and is unable to replicate past the lesion. Replication of damaged DNA templates can occur by error-free damage avoidance processes in which the undamaged complementary sequence is used to accomplish replication through the damaged site (14) or it may involve TLS by a specialized DNA polymerase across from the lesion. The S. cerevisiae RAD6 and RAD18 ...

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

“…Although the biochemical evidence is lacking, genetic evidence in yeast is consistent with a role of Rad5 acting as a ubiquitin ligase in Mms2-Ubc13-dependent polyubiquitylation of PCNA (17,19). All three Rad6-Rad18-dependent lesion bypass pathways are rendered inactive in the absence of PCNA ubiquitylation (19)(20)(21). Monoubiquitylation of PCNA is necessary for TLS by Pol and Po , and polyubiquitylated PCNA activates the Rad5-dependent PRR pathway.…”

“…Monoubiquitylation of PCNA is necessary for TLS by Pol and Po , and polyubiquitylated PCNA activates the Rad5-dependent PRR pathway. Because PRR becomes as highly defective in the K164R (pol30-119) mutant of PCNA as in the rad5⌬ and mms2⌬ mutants (20), PCNA polyubiquitylation is a necessary prerequisite for the activation of this PRR pathway.…”

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

“…Genetic data indicate that Srs2 negatively regulates recombination [70,71] possibly by reversal of intermediate recombination structures [72][73][74][75]. Indeed, the DNA strand exchange mediated by Rad51 is inhibited by Srs2 through disruption of the Rad51-ssDNA filaments [76,77], and it turns out that sumoylated PCNA has increased affinity for Srs2 [78,79] and represses the Rad52-dependent recombination pathway [80]. These observations collectively support the hypothesis that Srs2 serves as a molecular switch between homologous recombination and DDT [6], and further confirm that the sensor for this switch is the state of PCNA modification.…”

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.…”