The steady-state level and stability of TLS polymerase eta are cell cycle dependent in the yeast S. cerevisiae

Abstract

Polymerase eta (Pol eta) is a ubiquitous translesion DNA polymerase that is capable of bypassing UV-induced pyrimidine dimers in an error-free manner. However, this specialized polymerase is error prone when synthesizing through an undamaged DNA template. In Saccharomyces cerevisiae, both depletion and overproduction of Pol eta result in mutator phenotypes. Therefore, regulation of the cellular abundance of this enzyme is of particular interest. However, based on the investigation of variously tagged forms of Pol eta, mutually contradictory conclusions have been reached regarding the stability of this polymerase in yeast. Here, we optimized a protocol for the detection of untagged yeast Pol eta and established that the half-life of the native enzyme is 80 ± 14 min in asynchronously growing cultures. Experiments with synchronized cells indicated that the cellular abundance of this translesion polymerase changes throughout the cell cycle. Accordingly, we show that the stability of Pol eta, but not its mRNA level, is cell cycle stage dependent. The half-life of the polymerase is more than fourfold shorter in G1-arrested cells than in those at G2/M. Our results, in concert with previous data for Rev1, indicate that cell cycle regulation is a general property of Y family TLS polymerases in S. cerevisiae.

Functions of Ubiquitin and SUMO in DNA Replication and Replication Stress

“…The D. melanogaster homolog of TRIP, NOPO, is known to assemble K63-linked chains, possibly indicating a regulatory function of TRIP-mediated polymerase η modification as well, and interactions of both TRIP and NOPO with several Y-family polymerases suggest a conservation of the process ( Wallace et al, 2014 ). In budding yeast, polymerase η has been found to be ubiquitylated as well; however, the effects of this modification on protein stability remain controversial ( Parker et al, 2007 ; Skoneczna et al, 2007 ; Pabla et al, 2008 ; Plachta et al, 2015 ). Ubiquitin-mediated proteolysis also controls the levels of budding yeast TLS polymerase Rev1 along the cell cycle, thus limiting the bulk of its mutagenic activity to the G2/M phase ( Waters and Walker, 2006 ).…”

Regulation of the abundance of Y-family polymerases in the cell cycle of budding yeast in response to DNA damage

“…Previous findings that the TLS polymerases Pol eta and Rev1 are regulated in the cell cycle under stress-free conditions (Waters and Walker 2006;Plachta et al 2015) prompted us to further investigate the regulation of these polymerases in response to DNA damage. Since it has previously been shown that tagging of Pol eta with various epitopes differently affects the stability of this polymerase (Plachta et al 2015), we analyzed the levels of the native form of Pol eta in the current study. On the other hand, we studied the levels of a Rev1-ProA fusion protein, as an assay for immunodetection of the native form of Rev1 in yeast extracts has not yet been developed.…”

“…The band intensities were normalized to the intensities of the respective Pgk1 bands and to intensities at time 0′. b, e, h, k FACS data were used to monitor S-phase progression in cells arrested in G1-phase of the cell cycle (Plachta et al 2015;Waters and Walker 2006). Our results indicated that the levels of these proteins did not significantly increased when cells arrested with α factor in G1-phase were treated with UV radiation (10, 50, or 80 J/m 2 ) ( Fig.…”

Regulation of Mammalian DNA Replication via the Ubiquitin-Proteasome System

“…For example, both Rad30 (the S. cerevisiae homologue of POL-η) and Rev1 undergo cell cycle-dependent proteolysis to limit mutagenic activity, and in the case of Rad30, this is mediated via the SCF ubiquitin ligase with the F-box protein Ufo1 (Waters and Walker 2006; Skoneczna et al 2007; Plachta et al 2015). The second, and perhaps more important, mechanism restraining TLS activity is mediated through deubiquitylation of monoubiquitylated PCNA via the USP1 isopeptidase (Huang et al 2006).…”