Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA.

G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA.

Regulation of Ku-DNA association by Yku70 C-terminal tail and SUMO modification.

The Ku70-Ku80 ring complex encloses DNA ends to facilitate telomere maintenance and DNA break repair. Many studies focus on the ring-forming regions of subunits Ku70 and Ku80. Less is known about the Ku70 C-terminal tail, which lies outside the ring. Our results suggest that this region is responsible for dynamic sumoylation of Yku70 upon DNA association in budding yeast. Mutating a cluster of five lysines in this region largely eliminates Yku70 sumoylation. Chromatin immunoprecipitation analyses show that yku70 mutants with these lysines replaced by arginines exhibit reduced Ku-DNA association at both telomeres and internal DNA breaks. Consistent with this physical evidence, Yku70 sumoylation deficiency is associated with impaired ability to block DNA end resection and suppression of multiple defects caused by inefficient resection. Correlating with these, yku70 mutants with reduced sumoylation levels exhibit shorter telomeres, increased G overhang levels, and altered levels of non-homologous end joining. We also show that diminution of sumoylation does not affect Yku70 protein levels or its interactions with protein and RNA partners. These results suggest a model whereby Yku70 sumoylation upon DNA association strengthens Ku-DNA interaction to promote multiple functions of Ku.

Ku must load directly onto the chromosome end in order to mediate its telomeric functions.

The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding-defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance.

Ku DNA End-Binding Activity Promotes Repair Fidelity and Influences End-Processing During Nonhomologous End-Joining in Saccharomyces cerevisiae.

The Ku heterodimer acts centrally in nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSB). Saccharomyces cerevisiae Ku, like mammalian Ku, binds and recruits NHEJ factors to DSB ends. Consequently, NHEJ is virtually absent in yeast Ku null (yku70∆ or yku80∆) strains. Previously, we unexpectedly observed imprecise NHEJ proficiency in a yeast Ku mutant with impaired DNA end-binding (DEB). However, how DEB impairment supported imprecise NHEJ was unknown. Here, we found imprecise NHEJ proficiency to be a feature of a panel of DEB-impaired Ku mutants and that DEB impairment resulted in a deficiency in precise NHEJ. These results suggest that DEB-impaired Ku specifically promotes error-prone NHEJ. Epistasis analysis showed that classical NHEJ factors, as well as novel and previously characterized NHEJ-specific residues of Ku, are required for the distinct error-prone repair in a Ku DEB mutant. However, sequencing of repair junctions revealed that imprecise repair in Ku DEB mutants was almost exclusively characterized by small deletions, in contrast to the majority of insertions that define imprecise repair in wild-type strains. Notably, while sequencing indicated a lack of Pol4-dependent insertions at the site of repair, Pol2 exonuclease activity, which mediates small deletions in NHEJ, contributed to imprecise NHEJ in a Ku DEB mutant. The deletions were smaller than in Ku-independent microhomology-mediated end-joining (MMEJ) and were neither promoted by Mre11 nuclease activity nor Sae2 Thus, the quality of Ku's engagement at the DNA end influences end-processing during NHEJ and DEB impairment unmasks a Ku-dependent error-prone pathway of end-joining distinct from MMEJ.

Quorum sensing and multidrug transporters in Escherichia coli.

Previously, we found that the quorum sensing transcription factor SdiA up-regulates AcrAB. Others found that a 4-quinolone was a quorum-sensing signal in Pseudomonas aeruginosa. In Escherichia coli, there are at least three multidrug transporters (AcrAB/TolC, MdfA, and NorE) that exude fluoroquinolones. Here, we show that DeltaacrAB, tolC210, or DeltanorE mutants have the same growth rate as WT cells in exponential phase but grow to higher cell density in stationary phase. Overproduction of either pump caused cells to reach lower density. mdfA had no effect. Conditioned medium (CM) from cells overexpressing acrAB represses cell growth more than CM from WT cells. CM from pump mutant cells represses cell growth less than CM from WT cells. These results were not affected by the deletion of luxS, which synthesizes the quorum-sensing signal autoinducer 2 (AI-2). Expression of the rpoS gene encoding the stationary phase sigma factor is induced earlier in cells overexpressing acrAB and later in acrAB mutant cells. These results support a model in which a natural function of AcrAB/TolC and NorE is to export signals for cell-cell communication. Drugs exported by pumps may resemble communication molecules normally exuded.

A role for topoisomerase III in a recombination pathway alternative to RuvABC.

The physiological role of topoisomerase III is unclear for any organism. We show here that the removal of topoisomerase III in temperature sensitive topoisomerase IV mutants in Escherichia coli results in inviability at the permissive temperature. The removal of topoisomerase III has no effect on the accumulation of catenated intermediates of DNA replication, even when topoisomerase IV activity is removed. Either recQ or recA null mutations, but not helD null or lexA3, partially rescued the synthetic lethality of the double topoisomerase III/IV mutant, indicating a role for topoisomerase III in recombination. We find a bias against deleting the gene encoding topoisomerase III in ruvC53 or DeltaruvABC backgrounds compared with the isogenic wild-type strains. The topoisomerase III RuvC double mutants that can be constructed are five- to 10-fold more sensitive to UV irradiation and mitomycin C treatment and are twofold less efficient in transduction efficiency than ruvC53 mutants. The overexpression of ruvABC allows the construction of the topoisomerase III/IV double mutant. These data are consistent with a role for topoisomerase III in disentangling recombination intermediates as an alternative to RuvABC to maintain the stability of the genome.