We performed additional sequencing in the mutant strains, isolated in our previous study from conditions with the high level of UV-induced LHM within the reporter LYS2
gene placed into subtelomeric region of chromosome V (ref. 29
and ). Formation of subtelomeric ssDNA in these experiments was triggered by shifting cdc13-1
mutant yeast cells to non-permissive temperature 37°C, which lead to telomere uncapping followed by 5′→3′ resection. While the mutants were isolated based on lysine auxotrophy indicative of inactivation of the LYS2
function, we proposed that they carry additional mutations in the left subtelomeric region of chromosome V as well as in other subtelomeric regions. Additional targeted and genome-wide sequencing provided important information about the size and distribution of LHM regions as well as about density of mutations.
Density of mutagenic damage in ssDNA.
In the systems that we have developed, only a fraction of cells that have ssDNA in the region of the mutation reporter at the time of acute DNA damage have the potential for hypermutation. Therefore, the density of mutations in the hypermutable fraction was estimated based on the distribution of single and multiple mutant alleles (refs. 29
and Sup. Table 1
). Damage-induced LHM completely depended on error-prone TLS by Polζ (Rev3, Rev7) and on Rev1. Similar to other kinds of error-prone TLS, damage-induced LHM also depended on PCNA-K164 ubiquitylation. The DNA polymerase Polη (RAD30
) provides an error-free TLS pathway for the major UV-lesion cyclobutane dimers, that can compete with error-prone TLS by Polζ and Rev.43
Therefore damage-induced LHM could be further enhanced in the absence of Polη. However, in our initial study we did not detect a statistically significant change in the overall frequency of mutations in the reporters of ssDNA-associated mutagenesis when Polη was deleted.29
We note that the frequency of mutant alleles of a reporter gene in a population that contains cells with LHM spanning a reporter depends upon the mutation density within the LHM region, on the LHM fraction in the population as well as several other parameters. Thus direct measurement of mutation density by sequencing reporter ORFs provides more accurate estimate of LHM (discussed in refs. 29
). Therefore, we have sequenced LYS2
ORFs from 22 UV-induced lys2
mutants in the rad30
Δ derivative of the cdc13-1
strain with a subtelomeric LYS2
reporter on the left arm of chromosome V ( and Sup. Table 1
). We chose the subtelomeric LYS2
reporter because of the ORF is twice as large as the DSB-associated CAN1
-mutation reporter. The densities of mutations within the lys2
mutant ORFs were very similar between Rad+
Δ backgrounds. One explanation of this similarity is that the Polζ/Rev1-dependent error-prone TLS dominates in damaged ssDNA of wild type cells, while Polη error-free TLS operates only as a supplemental mechanism. The prevailing role of Polζ/Rev1 TLS may be associated with special conditions with checkpoint activation, which is characteristic for cells experiencing DSB or uncapped telomere. Both damage checkpoint and cell cycle controls have been implicated into regulation of TLS by specialized DNA polymerases.46
Importantly, a density of approximately one mutagenic UV-lesion per 3 kb in the LHM segment is close to the expected density of pyrimidine dimers in DNA of our treated yeast cells based on prior estimates.39,41
We conclude that the main source of UV-induced LHM is lack of repair rather than a higher density of UV-damage in ssDNA and that most lesions give rise to mutations via TLS.
Table 1. Multiple mutations and lys2 mutation spectra resulting from UV irradiation of cdc13-1 yeast strains.
Regions of damage-induced LHM can be large.
The mutation reporters used in our previous studies did not allow detection of damage-induced LHM regions greater than 4 kb. This would be sufficient for detecting simultaneous multiple mutations within nearly any yeast ORF because the vast majority of genes in this organism lack introns. However, most genes in higher eukaryotes are much longer due to the presence of introns. For example, the sizes of human genes range from several hundred nucleotides to more than a megabase, with a median around 25–30 kb.47,48
Thus LHM would need to span tens of kilobases to generate multiple mutations in the ORF of an average mammalian gene.
In order to determine the extent of the UV-induced LHM area we employed capillary ABI (Applied Biosystems, Foster City, CA), technology to sequence 30 kb regions adjacent to the left telomere of chromosome V, where subtelomeric LHM was observed in the LYS2
reporter. We examined twelve lys2
mutants of the Rad+
strain obtained in a previous study in reference 29
, after UV-irradiation of cdc13-1
cells arrested at non-permissive temperature (37°C). This condition inhibits telomere capping and results in formation of long ssDNA tails by way of 5′→3′ resection.49–51
For the no LHM control, the same region was sequenced from nine lys2
mutants isolated after UV-irradiation of the culture kept at permissive 23°C temperature, a condition in which ssDNA is not formed. Sequenced regions of each of the control strains contained only the single mutation in the LYS2
which gave rise to the Lys- phenotype selected in the experiment. In contrast, lys2
mutants isolated from UV-induced LHM conditions contained up to 11 mutations with tracts of multiple mutations spanning over 17 kb from the telomere ( and Sup. Tables 2 and 3
). The mutation density in the LHM regions was constant over telomere-proximal 12 kb and declined beyond that. Similar to our previous observation29
and in agreement with UV damage specificity,52
most mutations were base substitutions with a strong bias toward changes of pyrimidines in the strand that would be retained after 5′→3′ resection from the uncapped telomere. Thus, our results with extended sequencing are in agreement with LHM originating from damaged ssDNA formed by resection from the uncapped left telomere of chromosome V. Since UV damage is comparable for ssDNA and dsDNA53,54
we cannot distinguish between damage to ssDNA formed by resection versus damage to dsDNA right before resection. It is worth note that in the follow-up study of DSB associated LHM caused by methylmethane sulfonate (MMS), a mutagenic agent with ssDNA-specific spectrum, we found that MMS-induced LHM is associated with lesions in ssDNA rather than with damage occurring in dsDNA immediately before resection.45
Furthermore, the strand bias observed in our previous work29 as well as in the current study makes it unlikely that LHM could be due to long-term inhibition of repair in subtelomeric dsDNA persisting through the next round of DNA replication. Such a pathway would result in lys2
mutations originating from both DNA strands and thus a mutation spectrum lacking strand bias. Altogether, long-range sequencing demonstrated that the eukaryotic yeast cell is capable of restoring at least 15 kb of single-strand DNA containing over 10 mutagenic lesions to functional dsDNA with multiple mutations. This indicates that the area of damage-induced LHM can encompass an average size human gene and produce sets of mutations scattered over the entire ORF.
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 (more ...)
Genome-wide landscape of damage-induced mutations in cells with LHM.
In our previous study, UV-induced LHM was observed only at a subtelomeric LYS2
reporter but not in other LYS
genes scattered across the yeast genome.29
This indicated that the mutation load due to UV-induced mutagenesis in the rest of the genome is low. In order to verify this, we explored the genome-wide landscape of UV-induced mutations in several of the yeast clones that were isolated from the subtelomeric LHM experiments and used for long-range sequencing described in the previous section.
Recent advances in high-throughput whole genome sequencing have made it possible to use the entire genome as a reporter in studies of spontaneous and induced mutagenesis in a number of species.55–61
A reference sequence is created from the DNA of the cells that are closely related to the clones or tissues in which mutagenesis is explored. With the current level of technology, mutations can be identified (called) with confidence only in unique or moderately repeated parts of the genome. In the case of yeast this could be as much as 80–90% of the genome because rDNA repeats (about 10% of the genome) are excluded from the analysis and the rest of the reference sequence contains gaps at moderately repeated sequences. The sequence reads from individual clones are then aligned against the reference sequence and mutations are called using special software packages. As a last step in the identification of damage-induced mutations, all changes that are found in more than one clone are removed from the list. These identical mutations could result either from errors in the reference sequence or could arise during propagation of the population that was a source of the reference sequence.
We used the Illumina GAIIx (San Diego, CA) sequencing platform and CLC Genomics Workbench (GWB) 4.0 (CLC Bio, Katrinebjerg, Denmark) software to build the reference sequence of the strain DAG760 and to call mutations from Illumina reads of five clones isolated from the condition with UV-induced subtelomeric LHM (cdc13-1
-arrested at non-permissive 37°C) as well as from three control clones obtained after UV-mutagenesis of cdc13-1
cells that stopped at G1
after growth at permissive temperature 23°C (see Materials and Methods and Sup. Materials
). The clones were separated from the reference population of cells by 25–30 cell generations. Based on the recent measurements of genome-wide spontaneous mutations in non-mutagenized yeast, it is expected that each clone would contain at most 1–2 new mutations.57,59
We confirmed the expected low level of genome-wide incidence of detectable mutations in non-mutagenized yeast cultures using our sequencing tools and software. In agreement with published data there was only one new base substitution mutation detected in the genomes of six non-mutagenized clones separated by ~25 cell generations from the reference population. This contrasts with 16–38 new mutations found in each of the sequenced clones isolated after UV-mutagenesis ( and Sup. Table 4
). The accuracy of the reference sequence and mutation calling was confirmed by comparing changes identified within Illumina/CLC GWB mutation reports (Sup. Table 4
) with mutations in the left 30 kb subtelomeric region of chromosome V that were identified by conventional Sanger (ABI) sequencing (see above; total of 240 kb sequenced for eight isolates; Sup. Tables 2 and 3
). All 47 mutations in that region called in high-throughput Illumina sequencing were also identified by Sanger capillary (ABI) sequencing. Importantly, there were only three mutations identi- fied by capillary sequencing that were not called by Illumina (Sup. Table 3
). These mutations were actually present in the majority of reads, but were not called by the CLC GWB software due to the stringency of our mutation calling parameters. These three mutations represented minor categories within UV-induced spectra: two mutations were complex combination of base substitutions and indels and one was a simple indel. Thus, there was only a minimal discrepancy between the total of 240 kb sequence obtained by capillary ABI versus the corresponding Illumina generated sequence information.
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 (more ...)
The total numbers of UV-induced mutations in each genome was dramatically less than the number of damaged nucleotides expected for doses of UV used in our experiments (~6,000 lesions per genome based on refs. 39
). This indicates an overall high repair capacity across the genome as compared with the lack of repair resulting in LHM, associated with ssDNA formed at uncapped telomeres. In our experiments, cdc13-1
cells were held at non-permissive temperature for 6 h before UV-irradiation. In prior studies, ssDNA was detected 10–30 kb from telomeres in cdc13-1
cells arrested in G2
by shifting to non-permissive 37°C temperature for 6 h.49
Questions about continuity and size distribution for ssDNA regions created by 5′→3′ resection in this system have yet to be addressed. The resection rate in yeast was measured carefully only with site-specific DSBs, where the 5′→3′ DNA degradation proceeded at a rate of approximately 4 kb per hour.51
While conditions may differ for the resection at uncapped telomeres, this value leads to an estimate of around 25 kb of ssDNA formed by 5′→3′ resection in the cdc13-1
-arrested cells and provides an opportunity to address mutations in the subtelomeric vs. internal regions of the genome. In the absence of subtelomeric ssDNA formation, 76 out 84 (90%) UV-induced mutations were located in internal regions of chromosome (93% of the sequenced genome), while only 8 (10%) mutations mapped to subtelomeric regions (comprising the remaining 7% of the sequenced genome) (). In contrast, the fraction of subtelomeric mutations was 46–69% of all changes induced by UV in cells in which there was an opportunity for generation of subtelomeric ssDNA. Many of the mutations in subtelomeric regions were due to changes in the vicinity of the left telomere of chromosome V, where selection was applied to inactivation of the LYS2
reporter (). There were several clusters of 2–4 unselected mutations (a total of 16 mutations in clusters) in other subtelomeric regions of cdc13-1
-arrested cells, while there were no clusters in subtelomeric regions of the control G1
cells (p < 0.02 by two-tailed Fisher's exact test). This is consistent with the observation of multiple resected telomeres in populations of cdc13-1
We conclude that uncapping and resection occurs at multiple telomeres in a single cell. Moreover, multiple areas of damage induced-LHM, associated with regions of transient ssDNA, can occur within the same cell.
To address the genome-wide incidence of unselected mutations, the subtelomeric region adjacent to left telomere of chromosome V was excluded from further calculations, because it contained mutant lys2
alleles selected within our experimental design. Densities of UV-induced internal mutations in control as well as in G2
cells were in agreement with mutation frequencies of the LYS2
subtelomeric reporter in the absence of ssDNA formation.29
The densities of subtelomeric mutations in three control isolates did not show a statistically significant difference from that of internal UV-induced mutations. In contrast, the density of subtelomeric mutations in cdc13-1
arrested cells was approximately 16-fold greater than the mutation density at the internal regions of the same genomes, suggesting that several regions of LHM could be tolerated in the same cell (). The density of mutations in the left subtelomeric region of chromosome V, where initial lys2
mutations were selected, was 7-fold greater than the density of unselected UV-induced mutations in other subtelomeric regions (compare with
). The unselected subtelomeric mutation clusters were also shorter than those in the left subtelomeric region of chromosome V, to which mutation selection was applied. This could be explained by incomplete detection of mutations in moderately repeated segments that are often present in subtelomeric regions63
and/or by shorter stretches of ssDNA in the majority of subtelomeres. In order to verify that increased mutation density in subtelomeric regions was due to mutations induced by UV in ssDNA, we summarized data about bases mutated in the reference genome (). In agreement with the well established mutagen specificity of UV, the majority of mutations associated with model LHM reporters in the vicinity of uncapped telomere or DSB were identified as changes of pyrimidines in the ssDNA formed by resection (reviewed in ref. 29
and ). Thus, if increased density of unselected subtelomeric mutations is due to ssDNA generated by 5′→3′ resection in telomeres, the same kind of bias is expected. For the format of genome-wide analysis, it is important to note that the sequence and mutations throughout the entire chromosome are reported in the 5′ to 3′ direction of the top strand. Therefore, it would contain the actual sequence of ssDNA generated by resection from right telomeres and the sequence complementary to ssDNA retained after resection from left telomeres. As expected, there were more base substitutions in pyrimidines of the top strand reported in right subtelomeric regions of cdc13-1
arrested cells, while for the left telomeres there were more substitutions reported in purines of the top strand (; p < 0.02 by two-tailed Fisher's exact test). In summary, whole genome sequencing confirmed that density of UV-induced mutations in G2
cells is high in subtelomeric regions, while remaining at a baseline level throughout the rest of the genome. Importantly, based on incidence of unselected mutation clusters yeast cells are capable of tolerating multiple areas of UV-damaged ssDNA.
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 (more ...)