The genetic information of cellular organisms is encoded within double-strand DNA (dsDNA) genomes. Yet during DNA transactions such as replication, transcription, or recombination, the DNA exists transiently in single-strand form. It has long been appreciated that moieties in the nitrogenous bases of DNA are susceptible to damage, from both endogenous and exogenous sources, and that the double helical duplex structure can protect such moieties from chemical attack, due to Watson-Crick base pairing as well as base stacking 
. Conversely, single-strand DNA (ssDNA) is significantly more vulnerable to various forms of chemical modification. For example, spontaneous deamination of cytosine to uracil occurs at least 100-fold more rapidly in ssDNA than in dsDNA 
. Similarly, the alkylation of either N1 in adenine or N3 in cytosine, each of which would result in lesions that block replication, occurs much more readily in ssDNA 
. Spontaneous depurination and depyrimidination each occurs four-fold more rapidly in ssDNA than in dsDNA 
. In addition, it has been shown that transcription of DNA is associated with increased frequency of mutation and recombination, possibly due in part to the increased susceptibility of transient ssDNA (of the non-coding strand) to endogenous DNA damage 
. Finally, lesions formed in transient ssDNA during genome replication 
likely would have to be bypassed by translesion synthesis (TLS), which can be error-prone, i.e. mutagenic 
. Thus, regions of ssDNA are expected to be at greater risk of damage than dsDNA in vivo
, since there could be many agents that are not reactive against dsDNA, but are reactive enough to damage ssDNA.
Indeed, we showed previously that regions of ssDNA formed by resection at double-strand breaks (DSBs) 
or uncapped telomeres 
, as well as ssDNA within dysfunctional replication forks 
, were prone to increased mutagenesis. Damage-induced mutagenesis in such ssDNA can lead to the formation of clusters of multiple simultaneous mutations, with a mutation density comparable to the expected density of induced DNA lesions. For instance, methylmethanesulfonate (MMS)-induced clusters of multiple point mutations originated from methylated bases within ssDNA that was formed by resection from a DSB or in transient ssDNA formed during replication 
. The number of mutations within an individual cluster, generated as a single ssDNA-associated event, often exceeded the total number of mutations in the rest of the genome that were accumulated during approximately 25 generations of growth in the presence of MMS. Therefore, a mutagen that acts weakly on dsDNA is sufficiently reactive to induce mutation clusters in transiently formed ssDNA.
Strikingly, similar clusters of mutations were discovered in four types of malignant tumors 
. A considerable proportion of such mutation clusters clearly exhibited the signature of endogenous single-strand specific mutagens, namely APOBEC cytosine deaminases. Moreover, from 9% to 42% of all mutations in these tumors occurred at sequence motifs preferred by APOBEC enzymes, indicating that ssDNA-specific mutagenesis could be a significant contributor to carcinogenesis. It is therefore possible that other ssDNA-damaging agents, with properties that are not as well-understood as that of APOBEC enzymes, very well could contribute to mutagenesis also. Thus, it is crucial to identify such mutagens and to elucidate their mechanisms of action.
In order to make accurate assessments of the potential hazard posed by such ssDNA-damaging agents, we devised a subtelomeric triple reporter gene system in budding yeast as a facile means to identify and characterize ssDNA-specific mutagens. This reporter system takes advantage of the propensity of such mutagens to generate clusters of multiple point mutations spanning more than 10 kb, which can inactivate multiple closely-spaced reporter genes simultaneously. First, we validated the approach by expressing a modified human APOBEC3G in the reporter strains, resulting in clusters of mutations caused by deamination of cytosines in the ssDNA reporter region. We then used the reporter strains to characterize the mutagenicity of sulfites, a class of sulfur (IV) oxide compounds that is present in the environment (mainly due to combustion of fossil fuels 
) and in the food supply (>0.34% by weight of some foods consists of sulfites 
). Using a concentration of bisulfite (1%) that is similar to levels measured in foods, we confirmed that bisulfite is a very potent in vivo
deaminating agent that reacted specifically with cytosines in ssDNA to generate large clusters of mutations. But unlike uracil formed by enzymatic cytosine deamination, the main mutagenic lesion caused by sulfites (5,6-dihydrouracil-6-sulfonate) was refractory to excision by uracil-DNA N-glycosylase, and often was bypassed with the aid of an error-prone TLS polymerase. The reaction of either APOBEC3G or bisulfite with ssDNA each resulted in a distinctive strand-coordinated, multi-mutation signature, reminiscent of mutation clusters found in cancers 
. Our results further underscore the necessity of identifying ssDNA-specific mutagens and investigating the molecular mechanisms by which they act in vivo