Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition

DNA synthesis errors are a dual-edged sword. At a population level, accurate DNA replication maintains species identity, yet a small fraction of replication errors creates mutations that improve fitness and fuel evolution. At an individual level, DNA synthesis errors can be beneficial, e.g., by allowing a virus or microbe to survive in an adverse environment or by promoting affinity maturation of antibodies. Replication errors can also result in mutations that have deleterious consequences, cell death, or carcinogenesis. Because replication fidelity underpins so much biology, it has been intensively studied. These studies reveal that—in the absence of stress—replication fidelity is largely determined by nucleotide selectivity, proofreading, and mismatch repair (MMR), with considerable heterogeneity in each process (for review, see Kunkel 2009). Mutation rate heterogeneity is a feature of evolution (Sasaki et al. 2009; Prendergast and Semple 2011; Tolstorukov et al. 2011), including somatic evolution, i.e., tumorigenesis (for review, see Salk et al. 2010). This heterogeneity complicates the identification of genes responsible for the initiation and progression of cancer (Lawrence et al. 2013). Our understanding of the origins of heterogeneous replication fidelity is limited because most studies only monitor a tiny fraction of large, highly organized genomes. Whole-genome studies are required for a complete picture of variations in replication fidelity, the underlying mechanisms, and the consequences for evolution and disease. One way to interrogate global replication fidelity is to allow mutations to accumulate through many cell divisions with minimal selection against deleterious mutations, and then to sequence the genome to identify the types, numbers, and locations of the mutations that arise (Nishant et al. 2009). To focus on replication errors per se, rather than on other sources of spontaneous mutations, mutation accumulation can be studied in cells defective in nucleotide selectivity, proofreading, or MMR. Such studies have been done in Saccharomyces cerevisiae, whose haploid nuclear genome contains 16 chromosomes and 12 million base pairs (bp). Studies of strains with complete or partial defects in MMR reported the accumulation of 76 to 140 mutations, mostly deletions in homonucleotide runs (Zanders et al. 2010; Ma et al. 2012; Lang et al. 2013). Another study of MMR-deficient haploid yeast (Serero et al. 2014) reported 1679 mutations, mostly substitutions. We (Larrea et al. 2010) previously used an MMR-defective haploid strain encoding a mutator variant of DNA polymerase delta (Pol delta), one of three major nuclear replicases. From the genome-wide distribution of 1099 transitions that accumulated and from similar studies using a reporter gene (for review, see Kunkel and Burgers 2008; Lujan et al. 2013), we proposed a model wherein DNA polymerase alpha (Pol alpha) and Pol delta are primarily lagging-strand replicases, whereas polymerase epsilon (Pol epsilon) is primarily a leading-strand replicase. In these studies, small data sets and/or selective pressures precluded correlation of mutations with other key features of genomic structure. Here we report a study based on more than 40,000 mutations, accumulated in the absence of selective pressure, in diploid yeast encoding wild-type replicases or mutator variants of Pol alpha, delta, or epsilon, each either proficient or defective in MMR. The results allow calculations of single-base error rates per base pair per generation for replication across the yeast nuclear genome. They also permit genome-wide estimates of the efficiency of MMR for different mismatches. We find that fidelity varies with DNA sequence context, and establish relationships between fidelity and replication origins, replication timing, nucleosome positions, and protein coding potential.