Proteasomal regulation of the mutagenic translesion DNA polymerase, Saccharomyces cerevisiae Rev1

Abstract

Translesion DNA synthesis (TLS) functions as a tolerance mechanism for DNA damage at a potentially mutagenic cost. Three TLS polymerases (Pols) function to bypass DNA damage in Saccharomyces cerevisiae: Rev1, Pol ζ, a heterodimer of the Rev3 and Rev7 proteins, and Pol η (Rad30). Our lab has shown that S. cerevisiae Rev1 protein levels are under striking cell cycle regulation, being ~50-fold higher during G2/M than during G1 and much of S phase (Waters and Walker, 2006). REV1 transcript levels only vary ~3-fold in a similar cell cycle pattern, suggesting a posttranscriptional mechanism controls protein levels. Here, we show that the S. cerevisiae Rev1 protein is unstable during both the G1 and G2/M phases of the cell cycle, however the protein's half-life is shorter in G1 arrested cells than in G2/M arrested cells, indicating that the rate of proteolysis strongly contributes to Rev1's cell cycle regulation. In the presence of the proteasome inhibitor, MG132, the steady-state levels and half-life of Rev1 increase during G1 and G2/M. Through the use of a viable proteasome mutant, we confirm that the levels of Rev1 protein are dependent on proteasome-mediated degradation. The accumulation of higher migrating forms of Rev1 under certain conditions shows that the degradation of Rev1 is possibly directed through the addition of a polyubiquitination signal or another modification. These results support a model that proteasomal degradation acts as a regulatory system of mutagenic TLS mediated by Rev1.

Roadworks of DNA Damage Bypass during and after Replication

“…In support of the postreplicative nature of DNA damage bypass, the levels of yeast Rev1, which is regulated by proteasomal degradation, is high in G2/M cells (78,79). This possibly re‰ects that the TLS mechanism targets ssDNA gaps after bulk DNA replication.…”

The Ubiquitin-Proteasome System and DNA Repair

“…This spontaneous mutator phenotype has been verified to be primarily PRR-based and unrelated to the HR and NER pathways (McIntyre et al, 2006). Knock-out of Rev3, which encodes the catalytic subunit of Pol , in an Ump1-deficient yeast strain correlates with a dramatic decrease in the frequency of UV-induced and spontaneous mutations, thereby suggesting that Ump1 may serve as a negative regulator of Rev3 (Podlaska et al, 2003;McIntyre et al, 2006;Wiltrout & Walker, 2011). In contrast, the presence of Rad30, which encodes Pol , in an Ump1-deficient background is associated with increased UV-sensitivity but a decrease in the frequency of spontaneous mutations (Podlaska et al, 2003;McIntyre et al, 2006).…”

REV7 is required for anaphase-promoting complex-dependent ubiquitination and degradation of translesion DNA polymerase REV1

“…Exactly when, where and how REV1 could be targeted by APC-CDC20 and APC-CDH1 require further investigations. Since yeast REV1 also undergoes proteasomal degradation, 47,48,50 it will be of great interest to see whether degradation of yeast REV1 is also mediated by APC.…”

“…Since the degradation of yeast REV1 is mediated by ubiquitindependent proteolysis, 50 we next asked whether human REV1 is also ubiquitinated. To address this question, FLAG-REV1 and 6× histidine-tagged ubiquitin (His-Ub) were expressed in HEK293T cells.…”

“…This agrees with a recent study, which documents proteasomal degradation of yeast REV1. 50 Therefore, proteasome-dependent destruction is a conserved mechanism in eukaryotes for regulation of REV1. The stability of human REV1 has also been shown to be regulated by other proteins, such as Hsp90 and the Fanconi anemia core complex.…”