Hypermutability of Damaged Single-Strand DNA Formed at Double-Strand Breaks and Uncapped Telomeres in Yeast Saccharomyces cerevisiae

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Abstract
The major DNA repair pathways operate on damage in double-strand DNA because they use the intact strand as a template after damage removal. Therefore, lesions in transient single-strand stretches of chromosomal DNA are expected to be especially threatening to genome stability. To test this hypothesis, we designed systems in budding yeast that could generate many kilobases of persistent single-strand DNA next to double-strand breaks or uncapped telomeres. The systems allowed controlled restoration to the double-strand state after applying DNA damage. We found that lesions induced by UV-light and methyl methanesulfonate can be tolerated in long single-strand regions and are hypermutagenic. The hypermutability required PCNA monoubiquitination and was largely attributable to translesion synthesis by the error-prone DNA polymerase ζ. In support of multiple lesions in single-strand DNA being a source of hypermutability, analysis of the UV-induced mutants revealed strong strand-specific bias and unexpectedly high frequency of alleles with widely separated multiple mutations scattered over several kilobases. Hypermutability and multiple mutations associated with lesions in transient stretches of long single-strand DNA may be a source of carcinogenesis and provide selective advantage in adaptive evolution. A variety of error avoidance mechanisms assure low mutation rates across the genome. Genetic defects in DNA replication or repair can lead to genome-wide increase in mutation frequency that may result in cancer predisposition and genetic disease. Transient localized hypermutability drastically differs in its biological consequences from genome-wide mutators. Since genome-wide hypermutability can cause reduced fitness due to accumulation of dysfunctional alleles, mutators are under negative selection pressure. By contrast, there would be less selection against temporary hypermutability within limited genomic regions, suggesting a special role in adaptive evolution and carcinogenesis. Mechanisms of transient hypermutability are poorly understood. Long stretches of single-strand DNA have been implicated but not demonstrated as a source of localized transient hypermutability. Using sophisticated yeast genetic systems that we developed, we found that transient stretches of chromosomal single-strand DNA at double-strand breaks and that telomeres can tolerate multiple lesions and are highly prone to damage-induced mutations, including a very unusual class of widely spaced multiple mutations. The hypermutability relied on error prone translesion DNA synthesis. Our work demonstrates a simple in vivo mechanism for localized transient hypermutability extending over several kilobases that can result in widely spaced multiple mutations without severe mutation load in the rest of the genome.

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