A single-strand specific lesion drives MMS-induced hyper-mutability at a double-strand break in yeast
Introduction
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], [4] and may increase the likelihood of carcinogenesis [5], [6]. Somatic hyper-mutability 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 double-strand breaks (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 double-strand break (DSB) (herein referred to as the template strand because it provides the template for DNA synthesis associated with DSB repair as illustrated in Fig. 1) 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 single-strand DNA (ssDNA) because the template strand likely appears as a transient ssDNA intermediate in the processing of ends for DSB repair [8], [9]. 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 (Fig. 1a), it is also possible that lesions in dsDNA could give rise to strand-biased LHM (Fig. 1b), if the lesions were not repaired and the complementary strand was removed prior to completion of DSB repair (Fig. 1c and d). 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.
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], [12], [13], [14], [15]. There does not appear to be a difference in the kinds of lesions induced by UV in ssDNA or dsDNA [16], [17], as suggested by similar mutation spectra in ssDNA and dsDNA [8], [18]. However, as shown in Fig. 2, the distribution of lesions induced by MMS is different in ssDNA and dsDNA, both in vitro and within cells [14], [19], [20], [21], [22], [23]. 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.
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.
Section snippets
Yeast strains
All yeast strains were constructed from a strain isogenic to CG379 with the following common markers MATα ade5-1 his7-2 leu2-3,112 trp1-289 ura3Δ which was used in our previous study [8]. The “No-DSB”, “DSB-cen” and “DSB-tel” strains are illustrated in Fig. 3.
Measurement of mutation frequency
Procedures were similar to those in the previous study [8]. Briefly, yeast strains were grown with shaking in rich liquid media (YPDA) for approximately 16 h and then 1.5 ml culture was diluted into 50 ml fresh 2% galactose synthetic complete
MMS frequently causes multiple mutations in the vicinity of a repaired DSB
Previously we showed that the MMS- and UV-induced mutagenesis at the CAN1 gene, a reporter for forward mutations in yeast, was greatly increased if a DSB was created nearby prior to exposure to the mutagens [8]. As described in Fig. 3, the site-specific DSB is produced by galactose inducible I-SceI in URA3 (DSB-tel) or LYS2 (DSB-cen) that are close to a CAN1 reporter. The can1 mutation frequencies increased from around 10−5 and 10−4 in the entire population to as much as 3% and 7% of the cells
Mutability of MMS in double vs single-strand DNA
We have demonstrated that not only does the combination of DSB + DNA damage leads to high levels of LHM [8] but also that the LHM is primarily due to non-repairable lesions caused by MMS in ssDNA. This conclusion is based on the unique spectrum of mutations induced by MMS that is largely attributed to the single-strand specific 3-meC. The finding that MMS efficiently induces multiple mutations only in the ssDNA generated by a site-specific DSB but not in the absence of a DSB (Table 1) argues that
Implications
The unique pattern of MMS-induced multiple mutations for ssDNA involving multiple cytosines can help dissect the contribution of resection to repair of various kinds of induced DSBs in yeast. Along this line, alkylation damage could give rise to DSBs at clustered sites [10], [43] as well as lesions in the subsequent resected ends. Also, the DSB repair system that we employ may be useful in addressing the presence of endogenous alkylating agents and their potential for contributing to LHM [41],
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
We thank Dr. Shay Covo and other members of the lab for many helpful discussions. We are grateful to Drs. Julie Horton, Jana Stone, Steven Roberts and Thomas Kunkel for critical reading of the manuscript and helpful suggestions. This work was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Project ES065073 to M.A.R.).
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