Mutability and mutational spectrum of chromosome transmission fidelity genes

Abstract

It has been more than two decades since the original chromosome transmission fidelity (Ctf) screen of Saccharomyces cerevisiae was published. Since that time the spectrum of mutations known to cause Ctf and, more generally, chromosome instability (CIN) has expanded dramatically as a result of systematic screens across yeast mutant arrays. Here we describe a comprehensive summary of the original Ctf genetic screen and the cloning of the remaining complementation groups as efforts to expand our knowledge of the CIN gene repertoire and its mutability in a model eukaryote. At the time of the original screen, it was impossible to predict either the genes and processes that would be overrepresented in a pool of random mutants displaying a Ctf phenotype or what the entire set of genes potentially mutable to Ctf would be. We show that in a collection of 136 randomly selected Ctf mutants, >65% of mutants map to 13 genes, 12 of which are involved in sister chromatid cohesion and/or kinetochore function. Extensive screening of systematic mutant collections has shown that ~350 genes with functions as diverse as RNA processing and proteasomal activity mutate to cause a Ctf phenotype and at least 692 genes are required for faithful chromosome segregation. The enrichment of random Ctf alleles in only 13 of ~350 possible Ctf genes suggests that these genes are more easily mutable to cause genome instability than the others. These observations inform our understanding of recurring CIN mutations in human cancers where presumably random mutations are responsible for initiating the frequently observed CIN phenotype of tumors.

Fig. 1

Features of the chromosome fragment and Ctf assay. a Schematic of chromosome fragment construction as described in the main text (Spencer et al. 1990). Briefly a plasmid with a unique restriction between Y′ and unique sequences (inverted triangle) is digested and used to transform a haploid strain. In this example, recombination (indicated by large X) of the Y′ element (i) fuses the chromosome fragment plasmid to the telomere on the right arm and coordinate recombination of a unique pericentromeric site (ii) fuses the left arm of the chromosome to the plasmid-telomere moiety to generate the independent chromosome fragment (iii). Diagonal lines indicate large chromosomal distances. b Schematic of the Ctf assay. ade2-101 cells form red colonies (left) which are suppressed to a white color by SUP11 (center). Mutagenesis of a Ctf gene leads to red sectors in a white colony. The colony on the far right is half-sectored. The frequency of half-sectored colonies, which are generated by loss of the CF in the first cell division on the plate, can be used to quantify chromosome loss rates

Fig. 2

Genomic tiling array analysis of CTF2 and CTF9 alleles. Prediction scores across the TOF1 (a) and SMC3 (b) open reading frames in the composite of four CTF2 and three CTF9 alleles respectively (“s” numbers indicate isolate number). A polymorphism in the strain background (asterisk) is also visible in all CTF2/TOF1 alleles. c, d Summary of sequencing data for TOF1 in 11 CTF2 alleles (c) and for SMC3 in three CTF9 alleles (d). Normal protein sequence is schematized as blue for Tof1p and orange for Smc3p.The amino acid number and identity of each mutation is noted. Three TOF1 frameshift mutations are indicated with additional amino acids schematized in red. SMC3 point mutations are indicated in green

Fig. 3

A network of CIN-associated gene ontology terms. GO terms associated with ≥20 CIN genes were included and then filtered for redundant terms to retain the most genes while simplifying the network. Node size is set to number of genes annotated to a term and edge weight is set to the number of genes overlapping between terms. The figure encompasses >80% of the 692 CIN genes reported (Stirling et al. 2011). Blue node color indicates ≥2 genes from the 13 multimember Ctf complementation groups from Spencer et al. (1990) (CTF1–CTF12, CTF18) are annotated to that GO term. Darker blue indicates more genes annotated to that term (i.e., establishment of/mitotic sister chromatid cohesion—12 genes; DNA replication—6 genes; DNA repair, meiosis—4 genes; mitosis—3 genes; chromosome, replication fork protection complex, kinetochore—2 genes)

Fig. 4

Possible role of CIN in human tumors. CIN mutations may facilitate the evolutionary process underlying tumorigenesis. This concept is also illustrated for mutator phenotypes in the literature (Stratton et al. ; Loeb 2011). From left to right, a cellular mutational pathway is fed by intrinsic and extrinsic factors and can lead to a CIN mutation (blue diamond). CIN creates a mutant population in which oncogenic mutations driving proliferation are more likely to occur (red star). The proliferative phenotype and increased CIN facilitate variation (green star) that can help tumors respond to the environmental challenges (e.g., hypoxia, chemotherapy)