Dna2 Helicase/Nuclease Causes Replicative Fork Stalling and Double-strand Breaks in the Ribosomal DNA of Saccharomyces cerevisiae

We have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae and contributes to the shortened lifespan of dna2 mutants. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We show directly that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the early aging, hypomorphic dna2-2 helicase mutant. Deletion of FOB1, encoding the fork barrier protein, suppresses the elevated pausing and DSB formation, and represses initiation at rDNA ARSs. The dna2-2 mutation is synthetically lethal with {Delta}rrm3, encoding another DNA helicase involved in rDNA replication. It does not appear to be the case that the rDNA is the only determinant of genome stability during the yeast lifespan however since strains carrying deletion of all chromosomal rDNA but with all rDNA supplied on a plasmid, have decreased rather than increased lifespan. We conclude that the replication-associated defects that we can measure in the rDNA are symbolic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones

Microarray analysis of the in vivo sequence preferences of a minor groove binding drug

<p>Abstract</p> <p>Background</p> <p>Minor groove binding drugs (MGBDs) interact with DNA in a sequence-specific manner and can cause changes in gene expression at the level of transcription. They serve as valuable models for protein interactions with DNA and form an important class of antitumor, antiviral, antitrypanosomal and antibacterial drugs. There is a need to extend knowledge of the sequence requirements for MGBDs from <it>in vitro </it>DNA binding studies to living cells.</p> <p>Results</p> <p>Here we describe the use of microarray analysis to discover yeast genes that are affected by treatment with the MGBD berenil, thereby allowing the investigation of its sequence requirements for binding <it>in vivo</it>. A novel approach to sequence analysis allowed us to address hypotheses about genes that were directly or indirectly affected by drug binding. The results show that the sequence features of A/T richness and heteropolymeric character discovered by <it>in vitro </it>berenil binding studies are found upstream of genes hypothesized to be directly affected by berenil but not upstream of those hypothesized to be indirectly affected or those shown to be unaffected.</p> <p>Conclusion</p> <p>The data support the conclusion that effects of berenil on gene expression in yeast cells can be explained by sequence patterns discovered by <it>in vitro </it>binding experiments. The results shed light on the sequence and structural rules by which berenil binds to DNA and affects the transcriptional regulation of genes and contribute generally to the development of MGBDs as tools for basic and applied research.</p

Marianne Bronner-Fraser

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