Anc1, a protein associated with multiple transcription complexes, is involved in postreplication repair pathway in S. cerevisiae

Abstract

Yeast strains lacking Anc1, a member of the YEATS protein family, are sensitive to several DNA damaging agents. The YEATS family includes two human genes that are common fusion partners with MLL in human acute leukemias. Anc1 is a member of seven multi-protein complexes involved in transcription, and the damage sensitivity observed in anc1Delta cells is mirrored in strains deleted for some other non-essential members of several of these complexes. Here we show that ANC1 is in the same epistasis group as SRS2 and RAD5, members of the postreplication repair (PRR) pathway, but has additive or synergistic interactions with several other members of this pathway. Although PRR is traditionally divided into an "error-prone" and an "error-free" branch, ANC1 is not epistatic with all members of either established branch, and instead defines a new error-free branch of the PRR pathway. Like several genes involved in PRR, an intact ANC1 gene significantly suppresses spontaneous mutation rates, including the expansion of (CAG)(25) repeats. Anc1's role in the PRR pathway, as well as its role in suppressing triplet repeats, point to a possible mechanism for a protein of potential medical interest.

Figure 1. Cell cycle distribution of wildtype and anc1Δ asynchronous cells before and after MMS exposure.

A. WT cells, B. anc1Δ cells. When log-phase growing cells in YPD reached an OD(600) of 0.2, MMS was added to half of the cells at a concentration of 0.015%, and aliquots were removed at the indicated times to monitor cell cycle distribution by flow cytometry. Profiles of untreated cells are shown in red shading, and profiles of treated cells are shown with a blue trace.

Figure 2. Epistasis analysis of ANC1 in known DNA repair pathways.

Survival after chronic MMS treatment for: A. YPD gradient plate with maximum dose 0.03% MMS, B. YPD gradient plate with maximum dose 0.035% MMS, C. WT (▪), anc1Δ (▴), rad51Δ (▾), rad51anc1Δ (♦). D. WT (▪), anc1Δ (▴), rad54Δ (▾), rad54anc1Δ (♦), E. WT (▪), anc1Δ (▴), rad26Δ (▾), rad26anc1Δ (♦), F. WT (▪), anc1Δ (▴), rad5Δ (▾), rad5anc1Δ (♦). Log-phase cells were diluted and plated on freshly poured MMS-containing YPD-agar plates or onto control plates with no MMS. Colonies were allowed to grow at 30°C for 2–5 days before counting. At least two replicates were counted per trial. Serial dilution and gradient plate replicates available in Figure S2.

Figure 3. Epistasis analysis of ANC1 with PRR pathway members.

A. Genetic relationships within the PRR pathway in yeast. Epistasis was determined by sensitivity of mutants to damaging agents. srs2Δ only suppresses the damage sensitivity of rad5Δ, ubc13Δ and mms2Δ mutants (modified from Ulrich, 2006). Survival after chronic MMS treatment for: B. WT (▪), anc1Δ (▴), srs2Δ (▾), srs2anc1Δ (♦), C. WT (▪), anc1Δ (▴), mms2Δ (▾), mms2anc1Δ (♦) D. WT (▪), anc1Δ (▴), ubc13Δ (▾), ubc13anc1Δ (♦), E. WT (▪), anc1Δ (▴), rev3Δ (▾), rev3anc1Δ (♦) F. WT (▪), anc1Δ (▴), rad30Δ (▾), rad30anc1Δ (♦). We made several attempts to create a rad18anc1Δ strain for epistasis analysis, but were unable to produce the double mutant by either mating or recombination, even in the presence of a covering plasmid bearing an intact RAD18. Log-phase cells were diluted and plated on freshly poured MMS-containing YPD-agar plates or onto control plates with no MMS. Colonies were allowed to grow at 30°C for 2–5 days before counting. Results are average of at least 2 replicates, error bars = SD, except in F.; gradient plate replica for F. in Figure S2. We were unable to create a rad18anc1Δ double mutant by either mating or transformation, even with Rad18 expressed from a covering plasmid during transformation.

Figure 4. UV-induced point and frameshift mutagenesis and spontaneous mutagenesis.

A. Induced point mutagenesis in BY4741 background: wildtype, anc1Δ and rev3Δ cells were exposed to UV doses as indicated. Results are mean of two replicates, +/−SD. B. and C. Induced frameshift mutagenesis in CG379-A₁₂ and CG379-A₁₄ backgrounds: WT and anc1Δ frameshift reversions to a functional Lys2 gene. Cells were exposed to UV doses as indicated. Results are mean of two replicates, +/−SD. D. Spontaneous point mutagenesis, +/−SD, measured as described in Materials and Methods. E. Spontaneous expansion in (CAG)₂₅ repeats, +/−SD, measured as described in Materials and Methods.