SBF transcription factor complex positively regulates UV mutagenesis in Saccharomyces cerevisiae

Abstract

The collection of gene deletion mutants of Saccharomyces cerevisiae was used to screen for novel genes required for UV-induced mutagenesis. We found the SBF transcription factor (Swi4/Swi6 protein complex) to be required for wild-type levels of UV mutability in forward and reverse mutation assay. Expression of translesion polymerase ζ component Rev7 was identified as a target of SBFdependent regulation. KeywordsYeast; UV Radiation; Mutagenesis; Translesion synthesis; Transcriptional regulation Genetic instability resulting from enhanced mutagenesis is a severe consequence of exposing pro-and eukaryotic cells to DNA-damaging agents [1]. Especially for bulky adducts, such as UV-C induced pyrimidine dimers, point mutations opposite DNA lesions of reduced coding capacity mainly arise through active processes that ensure the completion of replication of a damaged template. In recent years, it became clear that high-fidelity replicative DNA polymerases are temporarily replaced at the lesion site by one of several translesion polymerases whose active sites can accommodate modified bases while lacking proofreading activity [2,3]. A concerted action of one polymerase inserting a base opposite a lesion together with another polymerase extending from the imperfectly matched primer/template junction may be required [4]. The ensuing damage bypass may be error-free or error-prone, depending on type of lesion and polymerases involved. Most of the enhanced mutability following UV radiation is generated by such error-prone translesion synthesis. The majority of UV-induced and spontaneous mutations are dependent on polymerase ζ, a complex of .Our present understanding of the events leading to mutagenic translesion synthesis also assigns a major role to proliferating cell nuclear antigen (PCNA). PCNA is subject to ubiquitination by the Rad6/Rad18 complex [8]. Monoubiquitinated PCNA appears to facilitate the switch from replicative to bypass polymerase [9,10] and thus, ubiquitin binding domains of bypass polymerases were found to be important for function [11][12][13][14]. Mutation of PCNA that prevents ubiquitination abolishes most of UV-induced mutability [15,16] Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The now commercially available collection of deletion mutants of non-essential yeast genes has previously been exploited to screen for mutants with enhanced spontaneous mutability and gross chromosomal rearrangements [28,29]. Here, we used this collection to identify novel genes required for UV-induced mutagenesis that may have been missed previously. The identical approach h...

Eukaryotic Translesion DNA Synthesis on the Leading and Lagging Strands: Unique Detours around the Same Obstacle

“…TLS pols were not identified near actively progressing replication forks in human cells, unlike all other components of the replisome, suggesting that TLS pols do not travel with progressing replication forks. , Indeed, pol η is distributed uniformly throughout the nucleus during unperturbed S-phase in human cells, in contrast to PCNA, which is concentrated into intranuclear foci at sites of DNA replication . Furthermore, the protein levels of TLS pols ζ and η in human and S. cerevisiae cells do not fluctuate significantly during the cell cycle nor in response to UV irradiation. ,− Collectively, this suggests that TLS on a UV-damaged Okazaki fragment most likely occurs after pol δ-mediated DNA synthesis has initiated from the nascent hybrid primer upstream (5′) of the offending damage.…”

The available SRL3 deletion strain of Saccharomyces cerevisiae contains a truncation of DNA damage tolerance protein Mms2: Implications for Srl3 and Mms2 functions

“…The commercially available collection of deletion mutants of non-essential genes of a haploid yeast strain (BY4741) was screened for strains with higher spontaneous mutation frequencies, using canavanine resistance as a forward mutation marker (Gong and Siede, 2009). While this screen was similar to the one described earlier by others (Huang et al, 2003), two novel genes were identified: SRL3 and TOP3 .…”

Lack of G1/S control destabilizes the yeast genome via replication stress-induced DSBs and illegitimate recombination

“…However, in swi6Δ cells, this repair pathway cannot be used effectively because DNA polymerase ζ does not function in these cells. The Rev7 protein, the regulatory subunit of DNA polymerase ζ, is barely detectable in swi6Δ (Gong and Siede, 2009). The shortage of the Rev7 protein and the resulting lack of DNA polymerase ζ activity are likely responsible for the extremely low mutation rate in swi6Δ (Fig.…”

“…We believe that the increased DNA rearrangement in cells lacking SWI6 is due to the accumulation of the recombinase Rad51 [a single-stranded (ss)DNA-binding protein responsible for homology searching during recombination (Sung, 1994)] and the diminished level of Srs2 (an Rad51 translocase), which together redirect DSB repair to an illegitimate recombination subpathway. This possibility is supported by the lower frequency of point mutations in the swi6Δ mutant than in the wild type (WT), likely due to Rev7 decay (Gong and Siede, 2009). Rev7 is a regulatory subunit of DNA polymerase ζ, which is responsible for most of the spontaneous mutations in haploid strains (Northam et al, 2010;Stone et al, 2012).…”