Localized somatic hyper-mutability is a phenomenon of accumulating multiple mutations in a small section of the genome. It may lead to a rapid increase in fitness (1
) or contribute to tumorigenesis (2
). Several lines of evidence suggest that the persistence of long stretches of single-stranded DNA (ssDNA) either in the vicinity of a double-strand break (DSB) or at uncoupled replication forks can lead to accumulation of such clustered mutations (2–6
). Both acute and chronic exposure to exogenous or endogenous mutagenic factors increase the incidence of mutations and amplify the probability of clustered, multiple genomic variations allowing for survival and further clonal expansion of malignant cells (7
). The inability of the majority of DNA repair systems to process lesions in single-stranded substrates is likely to increase the contribution of ssDNA damage to hyper-mutability. Therefore mechanisms and origins of spontaneous and induced mutagenesis ssDNA are of special interest.
The levels of spontaneous mutations in ssDNA can be orders of magnitude higher than in dsDNA (4
). Hyper-mutability may be caused by increased rate of errors during re-synthesis of the second DNA strand and/or by lesions inflicted by the products of cellular metabolism. Exposure to DNA-damaging agents (MMS, UV-irradiation and sulfites) further amplifies hyper-mutability of ssDNA (6
). Oxidative stress defines the shape of multiple biological processes since normal aerobic metabolism generates a highly reactive, oxidizing environment for all macromolecules in living organisms. Increased levels of reactive oxygen species (ROS) are implicated in the etiology and progression of pathological conditions such as cancer (10
), chronic inflammation (11
) and neurodegenerative diseases (12
). Oxidative stress is known to be a threat to genomic integrity (13–15
ROS damage DNA and produce dozens of chemically distinct lesions that distort the structure and coding properties of DNA and may cause mutations and chromosomal aberrations. Despite numerous reports on the correlation between elevated cellular levels of ROS and increased mutations and chromosome aberrations rates (14
), the precise mechanisms of ROS-induced genome destabilization remain poorly understood. Delineation of the pathways, contributing to ROS-induced genome instability is complicated by several factors including (i) redundancy and overlapping specificities of DNA damage repair and tolerance pathways (17
); (ii) the ability of the replication fork to bypass oxidative damage with relatively high efficiency (18
); and (iii) the absence of simple and reliable methods for detection and quantification of oxidative DNA damage in vivo
as well as the low sensitivity of existing methods for determining endogenous ROS levels.
The major confounding factor in defining the potential to generate mutagenic lesions and the mutagenic signature of both endogenous and exogenous oxidative damage appears to be the redundancy of pathways for repairing oxidative DNA damage. The majority of enzymes involved in the DNA base excision repair (BER) pathway, which is the major pathway for repair of oxidative DNA damage, have a broad spectrum of substrates and can replace each other at the initial steps of repair. Furthermore, when the capacity of BER is severely compromised, the nucleotide excision repair (NER) pathway, normally involved in processing of bulky DNA lesions can function in the repair of a sub-set of oxidative DNA lesions (17
). To make the picture even more complex, the post-replicative mismatch repair system can also recognize and remove 8-oxoG-A mis-pairs (20
). Therefore, defining an unaltered mutagenic signature of oxidative damage requires simultaneous elimination of multiple systems of protection acquired during the evolution of aerobic organisms. In principle, this can be achieved through analysis of the mutagenesis in strains in which multiple DNA damage-handling genes are compromised. Although such approaches has been used by several groups (21
) it is likely, that misinterpretation of the mutation signature data occurs due to the indirect effects of multiple mutations in the cellular background. For example, a complete elimination of both BER and NER pathways in yeast requires disruption of at least four DNA repair genes. Such quadruple mutants show significant changes in transcription patterns of different genes (23
), as compared to defects in either BER or NER caused by a single gene disruption. Such pleiotropic deregulation could significantly affect the mutation spectra, making it difficult to recapitulate the spectrum of oxidative mutagenic lesions in cells corrupted for both BER and NER. An alternative to inactivation of BER and NER would to utilize an ssDNA substrate that cannot be processed by the major DNA repair pathways even if those pathways are completely functional in the rest of the genome.
In this study we employed budding yeast strains with an ssDNA mutation reporter, which is not influenced by indirect effects and is free from ambiguity in determining the damaged nucleotide giving rise to a base substitution. This allowed generation of a more accurate picture of the mutagenic properties of ROS. We also assessed the relative contributions of oxidative stress to hyper-mutability and gross chromosomal rearrangements (GCRs) by measuring mutation frequencies and defining the signature of oxidative DNA damage in a sensitive ssDNA reporter system. Utilization of this tool allowed identification of mutagenesis-prone targets of oxidative damage and made it possible to compare the mutagenic impact of ROS on ssDNA to that of other types of DNA-damaging agents such as UV-irradiation, methyl methanesulfonate (MMS) and sulfites. We also addressed the role of translesion synthesis (TLS) polymerase zeta in oxidative stress-induced mutagenesis. Our results challenge several established views on mutagenesis in higher organisms and could have important implications for development of the therapeutic strategies aiming at selective killing of the cancer cells via increase in their mutational load.