The findings of these studies demonstrate the potent mutagenic effects of mirror repeat Pu·Py tracts both within the PKD1 Pu·Py tract itself as well as the surrounding sequence and the important role of specific helicases in preserving genetic stability at such DNA sequences (). The mutagenic potential of such tracts can be keenly dependent on the orientation encountered by the replication fork.
Model of RecQ and RecG helicases in replicative stress from triplex-forming sequences
Several members of the RecQ subfamily, specifically bacterial RecQ and mammalian WRN and BLM helicases, melt triplex [20
] and G-quartet (G4) DNA [19
]. We hypothesized that inactivation of RecQ helicases would accentuate mutagenesis surrounding Pu·Py tracts due to the increased replication blockade. We demonstrated an eight to tenfold increased risk of reversion of a triplex-forming plasmid in a RecQ-deficient background. Sequencing of these mutants show the overwhelming majority (>96%) involve inversion of the Pu·Py tract into the permissive orientation. Such an inversion event has been described in the inversion of the bacterial FimA3 promoter regulating fimbrial protein production [45
], acting as a genetic “on-off” switch. The inversion bias appears to be influenced by the degree of negative supercoiling [46
] and directed by recombinases such as HbiF, an invertase [47
]. This suggests that the inversion event that occurred in our system could be mediated by recombination, and stimulated by stabilized triplex formation in the absence of RecQ. This would be consistent with the hyperrecombination phenotype of RecQ deficiencies in humans, such as Werner and Bloom syndromes [23
RecG helicase participates in fork regression and replication restart reactions. Both BLM and WRN helicases have a similar fork regression activity [24
]. We did not find an increase in mutation frequency in sequence adjacent to triplex-forming Pu·Py tract in a RecG-deficient background, but we did find that loss of RecG helicase appeared to confer an approximately four fold decrease in mutation of the 88 bp tract itself as seen in previous studies [48
], suggesting that fork reversal by RecG may be an error-prone repair mechanism on repeated sequences. Loss of both RecG and RecQ caused an approximately three-fold increase in mutation frequency over that of wild type and RecQ-deficient cells of the 88bp tract, suggesting a synergism between both helicases. RecG did appear to be critical to maintaining stability of large Pu·Py tracts as evidenced by deletion of the entire 2.5kb tract in pCW31, but with the additional loss of RecQ, the efficiency of complete deletion was dampened suggesting that RecQ may be participating in the deletion of the large tract by its role as a component of the RecFOR recombination pathway.
Structure-mediated mutagenesis can be influenced by the orientation of the sequence. The expansion of (GAA·TTC)n
triplet repeat sequences within the FRDA
gene responsible for Friedreich’s ataxia has been ascribed to triplex formation in the nascent strand and pausing of replication machinery, leading to polymerase misalignment. [49
]. These triplet repeat sequences may also undergo deletion due to structure formation in the template strand [50
], absorbing negative superhelical tension [51
]. We found both complete and partial deletions when a Pu·Py tract was inserted into the CAT gene of pBR325. The two to three log order difference between orientations of the tract suggest that mutagenic triplex forms preferentially when the polypyrimidine sequence is in the leading strand, similar to hairpin formation studied by Sinden et al in this system [32
]. Furthermore, the nature of mutations as shown in Table 1 suggests that when triplex forms in this unidirectional plasmid in the leading strand, partial deletions occur. Loss of RecQ helicase did not appear to increase reversion rate above that of RecQ+
bacteria, but due to the nature of the assay out-of-frame deletions would remain chloramphenicol-sensitive and be undetected.
We detected a decreased expression of lambda repressor in the plasmid containing the Pu·Py tract in one orientation (pFLR), preventing its use in reversion analysis. This orientation-specific effect may be the result of the formation of replication-stalling structures by the tract, biasing the direction of replication of the pUC-based plasmid to preferentially create a “head-on” collision of the replication fork with elongating transcription machinery [52
]. This hypothesis is supported by the persistence of the effect on lambda repressor expression independent of distance from the promoter/lambda repressor cassette, and restoration of kanamycin sensitivity and thus lambda repressor expression with removal of the tract, as well as placing the sequence downstream of the repressor gene. An alternative explanation of this orientation-specific suppression of lambda repressor expression is the production of non-coding regulatory RNA (reviewed in reference [54
]) or the cryptic presence of a clustered regularly interspaced palindromic repeat (CRISPR) sequence [55
] from within adjacent pUC backbone. Deletion of the pUC backbone between the Pu·Py tract and the beta-lactamase gene of pFLR did not alter the orientation-specific effect (data not shown), suggesting that this possibility is unlikely. The expression of other plasmid-encoded proteins such as beta-lactamase (), plasmid copy number, methylation, and organism growth rates (data not shown) were also found to be similar between the different plasmids.
In summary, these studies suggest that RecG and RecQ serve distinct roles in maintaining replication fidelity and genomic stability of Pu·Py tracts. Mirror-repeat tracts may require RecQ activity to melt alternative DNA structures such as triplex to prevent fork blockade and sequence-mediated mutagenesis, but a series of these inverted repeats in tandem may provoke replication fork pausing and collapse, necessitating RecG activity to facilitate fork regression to maintain the stability of such large tracts.