Mutation spectrum data for Saccharomyces cerevisiae psf1-1 pol2-M644G mutants

DNA replication in Saccharomyces cerevisiae and other eukaryotes is performed mainly by polymerase epsilon (Pol ε) on the leading strand and polymerase delta (Pol δ) on the lagging strand. Using a mutant form of a DNA polymerase enables tracking its signature in the replicated DNA. Here, we used the pol2-M644G allele encoding the catalytic subunit of Pol ε to analyse its contribution to DNA replication in yeast with the psf1-1 allele of an essential gene encoding a subunit of the GINS complex. GINS is involved in the recruitment of Pol ε, the major leading strand replicase. Thus, its defective functioning can affect the involvement of Pol ε in DNA replication. Together with Cdc45 and Mcm2-7, GINS forms the CMG helicase complex. Our DNA sequencing data enable the observation of changes in the mutational spectra in the URA3 reporter gene cloned in two orientations regarding the nearest ARS. The data presented in this article support the study "Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex” [1].

a b s t r a c t DNA replication in Saccharomyces cerevisiae and other eukaryotes is performed mainly by polymerase epsilon (Pol ε) on the leading strand and polymerase delta (Pol δ) on the lagging strand. Using a mutant form of a DNA polymerase enables tracking its signature in the replicated DNA. Here, we used the pol2-M644G allele encoding the catalytic subunit of Pol ε to analyse its contribution to DNA replication in yeast with the psf1-1 allele of an essential gene encoding a subunit of the GINS complex. GINS is involved in the recruitment of Pol ε, the major leading strand replicase. Thus, its defective functioning can affect the involvement of Pol ε in DNA replication. Together with Cdc45 and Mcm2-7, GINS forms the CMG helicase complex. Our DNA sequencing data enable the observation of changes in the mutational spectra in the URA3 reporter gene cloned in two orientations regarding the nearest ARS. The data presented in this article support the study "Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex" [1] .

Value of the Data
• These are the first data on mutation specificity of the M644G-Pol2 variant alone and combined with the psf1-1 allele under conditions of polymerase zeta (Pol ζ ) inactivation ( REV3 deletion) and mismatch-repair mechanism deficiency ( MSH6 deletion). • These data can be beneficial for researchers deciphering DNA replication mechanisms and studying the fidelity of this process as well as the proteins involved in it. • These data provide information on the mutation spectra of the pol2-M644G in psf1-1 mutant cells.

Data Description
DNA replication in eukaryotic cells is conducted primarily by Pol ε and Pol δ on the leading and lagging strand, respectively. This division of labour may be changed under some conditions e.g. when the replication process is perturbed. Using specific mutants in genes encoding catalytic subunits of DNA polymerases, one can track the involvement of either replicase in DNA synthesis. The M644G variant of Pol2, the catalytic subunit of Pol ε demonstrates significantly higher rates of T • dT mispairs than A • dA [2] ( Fig. 1 ). Here, the signature of pol2-M644G was analysed in the psf1-1 mutant in the rev3 msh6 background. PSF1 encodes the psf1 essential subunit of the GINS complex, a component of the CMG helicase [3] . The psf1-1 subunit demonstrates impaired interaction with another GINS subunit -Psf3 [ 1 , 4 ], resulting in impaired functioning of the complex, what can affect the contribution of Pol ε to DNA replication. Deletion of REV3 inactivates DNA polymerase zeta (Pol ζ ) activity partially responsible for the increased mutation rates in the psf1-1 mutant [4] . The MSH6 gene was inactivated to impair the mismatch repair mechanism (MMR), correcting replication errors. The mutation spectra were analysed in the re- is cloned in two orientations (OR1 and OR2) in the vicinity of ARS306. Its coding sequence is replicated as the lagging strand in OR1 and as the leading strand in OR2. Therefore, T • T mispairs can be detected as A to T substitutions in URA3 OR1 and T to A in URA3 OR2.

Table 1
Mutation rates calculated for specific mutation types in the URA3 sequence. Data for control strains OR1, OR2, psf1-1 OR1, and psf1-1 OR2 are presented in the related research article [1] .
For mutation rate analyses, each of two or three independent isolates of each strain was used to inoculate at least eight cultures (2 ml each) grown at 23 °C until stationary phase. Then, appropriate dilutions of yeast cultures were plated on nonselective and selective (supplemented with 5-FOA for selection of URA3 mutants) media. After 4-7 days, yeast colonies were counted. To calculate spontaneous mutation rates, the μ = ƒ/ln(Nμ) equation was used [ μ -mutation rate per round of DNA replication; ƒ -mutant frequency (cell count from selective media divided by the cell count from nonselective media), and N -total population] [8] . Median values of mutation rates and 95% confidence intervals were calculated (GraphPad Prism software).
To characterize the mutation spectrum in the URA3 reporter gene, 103-338 5-FOA-resistant colonies were analyzed for each strain. Each colony represents an independent culture that was diluted and plated on a 5-FOA-containing SD medium. Primers URA3F393 (AACGAAGGAAGGAG-CACAGAC) and URA3R412 (CCGAAATTCCTGGGTAATAAC) were used to PCR-amplify the URA3 gene from 5-FOA resistant clones and for sequencing of the PCR product. The contribution of either mutation type to overall mutagenesis was calculated by dividing the number of specific events by the total number of mutations. Specific mutation rates were calculated proportionally to their contribution to the mutation spectrum.

Ethics Statements
This work involved neither human subjects, nor animal experiments, and adheres to Ethics in publishing standards.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.