ATP-dependent chromatin remodeling factors tune S phase checkpoint activity.

The S phase checkpoint response slows down replication in the presence of replication stress such that replication can resume normally once conditions are favorable. Both proper activation and deactivation of the checkpoint are crucial for genome stability. However, the mechanisms of checkpoint deactivation have been largely unknown. Here, we show that two highly conserved Saccharomyces cerevisiae ATP-dependent chromatin remodeling factors, Isw2 and Ino80, function to attenuate and deactivate S phase checkpoint activity. Genetic interactions revealed that these chromatin remodeling factors and the Rad53 phosphatases function in parallel in the DNA replication stress response. Following a transient replication stress, an isw2 nhp10 double mutant displays stronger and prolonged checkpoint activation without experiencing increased replication fork troubles. Isw2 and Ino80 are both enriched at stalled replication forks and physically and specifically interact with a single-stranded DNA binding protein, replication protein A (RPA). Based on these results, we propose that Isw2 and Ino80 are targeted to stalled replication forks via RPA and directly control the amplitude of S phase checkpoint activity and the subsequent deactivation process.

Coprinus cinereus rad50 mutants reveal an essential structural role for Rad50 in axial element and synaptonemal complex formation, homolog pairing and meiotic recombination.

The Mre11/Rad50/Nbs1 (MRN) complex is required for eukaryotic DNA double-strand break (DSB) repair and meiotic recombination. We cloned the Coprinus cinereus rad50 gene and showed that it corresponds to the complementation group previously named rad12, identified mutations in 15 rad50 alleles, and mapped two of the mutations onto molecular models of Rad50 structure. We found that C. cinereus rad50 and mre11 mutants arrest in meiosis and that this arrest is Spo11 dependent. In addition, some rad50 alleles form inducible, Spo11-dependent Rad51 foci and therefore must be forming meiotic DSBs. Thus, we think it likely that arrest in both mre11-1 and the collection of rad50 mutants is the result of unrepaired or improperly processed DSBs in the genome and that Rad50 and Mre11 are dispensable in C. cinereus for DSB formation, but required for appropriate DSB processing. We found that the ability of rad50 mutant strains to form Rad51 foci correlates with their ability to promote synaptonemal complex formation and with levels of stable meiotic pairing and that partial pairing, recombination initiation, and synapsis occur in the absence of wild-type Rad50 catalytic domains. Examination of single- and double-mutant strains showed that a spo11 mutation that prevents DSB formation enhances axial element (AE) formation for rad50-4, an allele predicted to encode a protein with intact hook region and hook-proximal coiled coils, but not for rad50-1, an allele predicted to encode a severely truncated protein, or for rad50-5, which encodes a protein whose hook-proximal coiled-coil region is disrupted. Therefore, Rad50 has an essential structural role in the formation of AEs, separate from the DSB-processing activity of the MRN complex.

ATP-dependent chromatin remodeling shapes the DNA replication landscape.

The eukaryotic DNA replication machinery must traverse every nucleosome in the genome during S phase. As nucleosomes are generally inhibitory to DNA-dependent processes, chromatin structure must undergo extensive reorganization to facilitate DNA synthesis. However, the identity of chromatin-remodeling factors involved in replication and how they affect DNA synthesis is largely unknown. Here we show that two highly conserved ATP-dependent chromatin-remodeling complexes in Saccharomyces cerevisiae, Isw2 and Ino80, function in parallel to promote replication fork progression. As a result, Isw2 and Ino80 have especially important roles for replication of late-replicating regions during periods of replication stress. Both Isw2 and Ino80 complexes are enriched at sites of replication, suggesting that these complexes act directly to promote fork progression. These findings identify ATP-dependent chromatin-remodeling complexes that promote DNA replication and define a specific stage of replication that requires remodeling for normal function.

Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA.

We show that intracellular transcription of G-rich regions produces novel DNA structures, visible by electron microscopy as large (150-500 bp) loops. These G-loops are formed cotranscriptionally, and they contain G4 DNA on one strand and a stable RNA/DNA hybrid on the other. G-loop formation requires a G-rich nontemplate strand and reflects the unusual stability of the rG/dC base pair. G-loops and G4 DNA form efficiently within plasmid genomes transcribed in vitro or in Escherichia coli. These results establish that G4 DNA can form in vivo, a finding with implications for stability and maintenance of all G-rich genomic regions.