Mutagenesis is one of the major driving forces of evolution and also contributes to carcinogenesis and genetic diseases in humans [1
]. Continuous strong genome-wide mutability can be deleterious to species [2
], while transient localized hyper-mutability (LHM) can provide opportunities for evolution without significantly increasing the load on fitness [3
] and may increase the likelihood of carcinogenesis [5
]. Somatic hypermutability in the immunoglobulin genes is a dramatic example of LHM that actually benefits an organism [7
Recently, using the yeast Saccharomyces cerevisiae
we reported that DNA damage can induce high levels of mutability in the regions near DSBs or at uncapped telomeres, providing new insights into mechanisms of LHM [8
]. The UV-induced LHM in a reporter gene exhibited strand-biased mutations toward changes of pyrimidines in the unresected strand used for recombinational repair of a DSB (herein referred to as the template strand
because it provides the template for DNA synthesis associated with DSB-repair as illustrated in ) as well as in the unresected strand that can arise transiently at uncapped telomeres. Therefore, the source of LHM was attributed to premutational lesions in ssDNA because the template strand likely appears as a transient ssDNA intermediate in the processing of ends for DSB-repair [8
]. Since the ssDNA would not be subject to excision repair, lesions would have a much greater potential for mutation than if they occur in dsDNA. Although the earlier results can be explained by damage in ssDNA (), it is also possible that lesions in dsDNA could give rise to strand-biased LHM (), if the lesions were not repaired and the complementary strand was removed prior to completion of DSB-repair (). Conversion of damaged dsDNA to damaged ssDNA prior to DSB-repair might occur, for example, in the case of an agent such as methyl methanesulfonate (MMS) that can generate clustered lesions in dsDNA leading to DSBs as well as cause single base damage in dsDNA and ssDNA [10
]. Damage that is specific to ssDNA can be instrumental in assessing the relative contribution of ssDNA vs
dsDNA to LHM in the vicinity of a DSB, as well as the density of lesions in ssDNA.
Lesions in ssDNA as well as unrepaired lesions in dsDNA can lead to strand-biased LHM around a repaired DSB
While the lesions produced by UV are mostly pyrimidine dimers or 6-4 photoproducts, MMS primarily induces single-base damage. Repair of either UV or MMS lesions involves excision and replacement of damaged nucleotides using the complementary strand as a template. Unlike for UV, an abasic intermediate is generated during base excision repair (BER) of MMS lesions. Removal of MMS damage from dsDNA as well as ssDNA also might occur through enzymatic reversal (e.g., E. coli
AlkB or its human homologues)[11
]. There does not appear to be a difference in the kinds of lesions induced by UV in ssDNA or dsDNA [16
], as suggested by similar mutation spectra in ssDNA and dsDNA [8
]. However, as shown in , the distribution of lesions induced by MMS are different in ssDNA and dsDNA, both in vitro
and within cells [14
]. We, therefore, anticipated that mutational spectra might reveal marked differences in the in vivo
mutational properties of MMS-induced lesions in ssDNA as compared to dsDNA.
Frequency (%) of MMS-induced base lesions among all dsDNA or ssDNA lesions
In the present study we have found that MMS actually generates a class of lesions that lead to mutations specific to ssDNA and that the overall lesion generation in ssDNA may be much greater than in dsDNA, suggesting that ssDNA is much more vulnerable than dsDNA to alkylation damage and subsequent genome instability. The nearly 20,000-fold difference in the density of MMS-induced mutations associated with DSB-repair regions, as compared to no DSB, primarily results from a combination of increased induction of lesions in ssDNA, induction of lesions that are more mutagenic and lack of repair of single-strand damage prior to DSB-repair.