The critical mutagenic translesion DNA polymerase Rev1 is highly expressed during G2/M phase rather than S phase -- Waters and Walker 103 (24): 8971 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Waters and Walker 10.1073/pnas.0510167103.

Supporting Information

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Materials and Methods

Fig. 5. The C-terminally tagged Rev1 construct retains full function. (A and B) Isogenic strains of the WT background, rev1D, and the tagged Rev1 were compared for survival (A) and mutagenesis (B) after 10 J/m² UV irradiation. The data plotted for the rev1D reversion frequency is the limit of detection, because no revertants were recovered. Therefore, the rev1D reversion frequency is likely lower than indicated.

Fig. 6. Rev1 is high throughout mitosis and is posttranscriptionally regulated. (A and B) Cells from the 60- (A) and 90-min (B) time points stained with DAPI and anti-tubulin to visualize DNA or anaphase spindles, respectively. (C) Immunoblot quantitating the change in Rev1 protein levels between minimal and maximal points; comparison of an undiluted G₁ sample to various dilutions (1- to 8-, 10-, 13-, 15-, 16-, 18-, 20-, 23-, 25-, 32-, 40-, 45-, and 50-fold) of a G₂/M sample. (D) RT-PCR samples of minimal and maximal REV1 mRNA levels were compared quantitatively by using 2-fold serial dilutions of input cDNA.

Fig. 7. Synchronization by elutriation shows Rev1 cell cycle expression. (A) Immunoblot against the protein A epitope tag of Rev1 and the loading control PGK (phosphoglycerate kinase). Small, unbudded cells were collected by centrifugal elutriation, and samples were taken at the indicated times after resuspension in rich media. (B) FACS analysis of the DNA content. (C) Plot showing quantitation of the immunoblot from A normalized in arbitrary units to a standard dilution curve.

Fig. 8. Rev1 may function after replication has been completed to fill gaps that remain opposite lesions. (A) Putative evolutionary conservation of delay in mutagenic DNA lesion processing. (B) Model showing generation of single-stranded DNA gaps in S phase by replication fork uncoupling at the site of a DNA lesion. Rev1 may recognize a persistent gap in G₂ and carry out limited TLS itself and/or recruit other factors, such as the other TLS polymerases.

Supporting Materials and Methods

Immunofluorescence.
Immunofluorescence was performed by using anti-tubulin (Oxford Biotechnology, Kidlington, Oxfordshire, U.K.) and anti-rat FITC (Jackson ImmunoResearch) (1). RT-PCR.
RNA was isolated by using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. To generate cDNA, 0.5 mg of RNA was used in 20-ml reactions with 20 pmol of oligo(dT) primer and 25 units of Stratascript reverse transcriptase (Stratagene). All PCRs were performed for 26 cycles, and different amounts of input cDNA were used to analyze relative levels of REV1 or ACT1 mRNA in each sample. Survival and Mutagenesis Assays
. Three independent cultures of each strain were grown to saturation, and appropriately diluted aliquots were plated on synthetic complete (SC) media to monitor survival. Mutation frequencies were analyzed by plating undiluted aliquots on SC-Trp media to score for reversion of the trp1-1 allele. Plates were irradiated at 1 J/m² per sec by using a G15T8 UV lamp (General Electric) at 254 nm and grown for 3 or 6 days at 30°C for survival and mutagenesis assays, respectively. Elutriation
. Cells were collected by elutriation as described in ref. 2 in a JE-5.0 Elutriator (Beckman Coulter) and resuspended in yeast extract/peptone/dextrose (YPD) media to initiate the time course.

1. Kilmartin, J. V. & Adams, A. E. (1984) J. Cell Biol. 98, 922-933.

2. Amon, A. (2002) Methods Enzymol. 351, 457-467.