Damage-induced localized hypermutability

Abstract

Genome instability continuously presents perils of cancer, genetic disease and death of a cell or an organism. At the same time, it provides for genome plasticity that is essential for development and evolution. We address here the genome instability confined to a small fraction of DNA adjacent to free DNA ends at uncapped telomeres and double-strand breaks. We found that budding yeast cells can tolerate nearly 20 kilobase regions of subtelomeric single-strand DNA that contain multiple UV-damaged nucleotides. During restoration to the double-strand state, multiple mutations are generated by error-prone translesion synthesis. Genome-wide sequencing demonstrated that multiple regions of damage-induced localized hypermutability can be tolerated, which leads to the simultaneous appearance of multiple mutation clusters in the genomes of UV- irradiated cells. High multiplicity and density of mutations suggest that this novel form of genome instability may play significant roles in generating new alleles for evolutionary selection as well as in the incidence of cancer and genetic disease.

Figure 1

Mutation frequencies associated with various kinds of localized hypermutability. Mutation frequencies were derived from the following publications: DSB-repair, F′ episome-E. coli mutations selected in F′ codA,B genes with a mutation target size of 848 bp was used for calculations; Double-stranded (ds) gap repair—mutations in the yeast URA3 gene occurring during a double-stranded (ds) gap repiair, a minimal estimate of mutation target of 125 bp as determined by; DSB-repair—frequency of spontaneous mutations in the vicinity of a DsB repaired by homologous recombination. A site-specific DsB was induced next to the chromosomal CAN1 gene and repaired by homologous recombination with a truncated copy of CAN1 in the same chromosome. Unlike a double-strand gap in reference 30, DSB ends homologous to adjacent segments of DNA sequence were used for repair. this system did not allow distinction between hypermutability in transiently formed ssDNA (as in ref. 29), or hypermutability during repair of a ds-gap (as in ref. 30). DSB-repair in ssDNA—in this system the CAN1 reporter gene was placed in the vicinity of a DSB that was repaired by a short oligonucleotide, so the CAN1 sequence did not participate in recombination and the most likely hypermutable intermediate was transient ss DNA (ref. and Fig. 2A). The probability of CAN1 mutations in both DSB-repair systems was calculated based on the frequency data corrected for the minimal estimate of mutation target (236 bp as determined by ref. 85). Damage-induced LHM (ssDNA at DSB or uncapped telomere). Average frequencies of mutations induced by Uv-C (45 J/m²) and MMS (30 min in 11.8 mM (0.1%) MMS) in yeast ssDNA around DsB or next to uncapped telomere were taken from references and . SHM, adaptive immunity. Approximate frequency of mutations associated with somatic hypermutation in the Ig-genes was taken from references and .

Figure 2

Damage-induced localized hypermutability associated with transient regions of single-strand DNA generated at double-strand breaks and uncapped telomeres. The experimental approach and conclusions summarized in this figure were described in references and . (A) Double-strand break (DSB). Long ssDNA can be generated around a DsB by 5′→3′ resection if DSB repair is delayed. While damage in dsDNA (gray stars) can be repaired by major repair pathways, such as base excision repair, nucleotide excision repair, post-replication repair, damage in ssDNA (yellow stars) often remains unrepaired. Repair of an inducible site-specific DSB was triggered by adding oligonucleotides complementary to the ends of the break. Trans-lesion DNA synthesis (TLS) is required to create a complementary strand on the damaged DNA template. The TLS events can generate wild-type sequence; however, if TLS is error-prone mutations (blue boxes) can be created at many DNA damage sites. (B) Uncapped telomere. Long ssDNA can be generated by 5′→3′ resection at telomeres that transiently lost their capping protein complex. This uncapping was achieved by shifting a cdc13-1 mutant to a non-permissive temperature (37°C). Restoration of the telomere cap and dsDNA was allowed by shifting back to permissive temperature (23°C) after applying DNA damage (Uv or MMS). Multiple mutations were generated by error-prone TLS as in (A). Mutations were initially detected in a reporter LYS2 gene (not shown), placed at 2.2 kb from left telomere of the chromosome V, based on lysine auxotrophy. Mutations in the other genomic areas of these lysine auxotrophs were identified in the current study by extended sequencing of the chromosome V subtelomeric region and later by whole genome sequencing (see Results).

Figure 3

UV-induced mutations in a subtelomeric area. (A) Presented are mutations in a 30 kb terminal region of the truncated left telomere region of chromosome 5 (details of the construction are described in ref. 29) in twelve lys2 mutants induced by UV-light in G₂ arrested at 37°C cdc13-1 cells. Not shown are nine lys2 mutants induced by UV-light in control G₁ stationary cells that were kept at permissive temperature (23°C). Each of nine mutants from control set contained only one lys2 mutation across the sequenced 30 kb region (Sup. Table 1). Each horizontal line under the map of the region corresponds to the sequence of an individual mutant. Symbols correspond to a nucleotide change in the single strand that would remain after resection of the complementary strand. See Supplemental Tables 2 and 3 for complete information about all identified mutations. (B) The average mutation densities (numbers above top grey lines) and total numbers of different mutation types (graph) in the 12 sequenced lys2 mutants originating from G₂-arrested cdc13-1 cells treated by Uv-light. All mutants were picked by inactivation of LYS2 function; therefore calculation of mutation density in the LYS2 segment (number in brackets) was based on the formula and assumptions described in footnotes for Table 1. The average mutation densities in the other two regions, where all mutations were unselected, were calculated as the total number of mutations divided by the total number of nucleotides sequenced in these regions.

Figure 4

Mutations identified by whole-genome sequencing in the individual genomes of UV-irradiated yeast cells. (A) Distribution of UV-induced mutations between subtelomeric and internal regions of yeast chromosomes. Subtelomeric regions were defined as 25 kb from either end of the reference chromosome sequence (see text). Shown are the total numbers of simple base substitutions, indels and complex changes for each type of regions. All mutations are listed in Supplemental Table 4. (B) Numbers of mutations in subtelomeric regions of each chromosome. (ss) indicates mutants obtained from cdc13-1 cells arrested in G₂ at 37°C, a condition triggering formation of ssDNA at uncapped telomeres; (ds) indicates mutants obtained from control G₁ stationary cdc13-1 cells at 23°C; under these conditions formation of subtelomeric ssDNA is not expected.

Figure 5

Density of unselected mutations and purine/pyrimidine bias in subtelomeric and internal regions of yeast chromosomes revealed by whole genome sequencing. The letter “m” precedes the number of specific mutant strains. In order to obtain parameters for unselected mutations, all changes in the 25 kb left subtelomeric region of chromosome V containing the LYS2 mutation reporter were excluded from calculations. (A) Density of mutations calculated for the total of base substitutions, indels and complex changes. (B) Purine/pyrimidine bias of simple base substitutions. Mutations were categorized based on nucleotide changes in the 5′→3′ (top) strand throughout the chromosome reference sequence.