Cohesin residency determines chromatin loop patterns.

The organization of chromatin into higher order structures is essential for chromosome segregation, the repair of DNA-damage, and the regulation of gene expression. Using Micro-C XL to detect chromosomal interactions, we observed the pervasive presence of cohesin-dependent loops with defined positions throughout the genome of budding yeast, as seen in mammalian cells. In early S phase, cohesin stably binds to cohesin associated regions (CARs) genome-wide. Subsequently, positioned loops accumulate with CARs at the bases of the loops. Cohesin regulators Wpl1 and Pds5 alter the levels and distribution of cohesin at CARs, changing the pattern of positioned loops. From these observations, we propose that cohesin with loop extrusion activity is stopped by preexisting CAR-bound cohesins, generating positioned loops. The patterns of loops observed in a population of wild-type and mutant cells can be explained by this mechanism, coupled with a heterogeneous residency of cohesin at CARs in individual cells.

The spatial regulation of condensin activity in chromosome condensation.

Condensin mediates chromosome condensation, which is essential for proper chromosome segregation during mitosis. Prior to anaphase of budding yeast, the ribosomal DNA (RDN) condenses to a thin loop that is distinct from the rest of the chromosomes. We provide evidence that the establishment and maintenance of this RDN condensation requires the regulation of condensin by Cdc5p (polo) kinase. We show that Cdc5p is recruited to the site of condensin binding in the RDN by cohesin, a complex related to condensin. Cdc5p and cohesin prevent condensin from misfolding the RDN into an irreversibly decondensed state. From these and other observations, we propose that the spatial regulation of Cdc5p by cohesin modulates condensin activity to ensure proper RDN folding into a thin loop. This mechanism may be evolutionarily conserved, promoting the thinly condensed constrictions that occur at centromeres and RDN of mitotic chromosomes in plants and animals.

Genome-wide Map of R-Loop-Induced Damage Reveals How a Subset of R-Loops Contributes to Genomic Instability.

DNA-RNA hybrids associated with R-loops promote DNA damage and genomic instability. The capacity of hybrids at different genomic sites to cause DNA damage was not known, and the mechanisms leading from hybrid to damage were poorly understood. Here, we adopt a new strategy to map and characterize the sites of hybrid-induced damage genome-wide in budding yeast. We show that hybrid removal is essential for life because persistent hybrids cause irreparable DNA damage and cell death. We identify that a subset of hybrids is prone to cause damage, and the chromosomal context of hybrids dramatically impacts their ability to induce damage. Furthermore, persistent hybrids affect the repair pathway, generating large regions of single-stranded DNA (ssDNA) by two distinct mechanisms, likely resection and re-replication. These damaged regions may act as potential precursors to gross chromosomal rearrangements like deletions and duplications that are associated with R-loops and cancers.

S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation.

R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae Using this methodology, we identified ∼800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast.

The Yin and Yang of R-loop biology.

RNA performs diverse functions in cells, directing translation, modulating transcription and catalyzing enzymatic reactions. Remarkably RNA can also anneal to its genomic template co- or post-transcriptionally to generate an RNA-DNA hybrid and a displaced single-stranded DNA. These unusual nucleic acid structures are called R-loops. Studies in the last decades concentrated on the detrimental effects of R-loop formation, particularly on genome stability. In fact, R-loops are thought to play a role in several human diseases like cancer and neurodegenerative syndromes. But recent data has revealed that R-loops can also have a positive impact on cell processes, like regulating gene expression, chromosome structure and DNA repair. Here we summarize our current understanding of the formation and dissolution of R-loops, and discuss their negative and positive impact on genome structure and function.

Break-induced replication repair of damaged forks induces genomic duplications in human cells.

In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.

An induced fit mechanism regulates p53 DNA binding kinetics to confer sequence specificity.

The p53 tumour suppressor gene, the most frequently mutated gene in human cancer, encodes a transcription factor that contains sequence-specific DNA binding and homo-tetramerization domains. Interestingly, the affinities of p53 for specific and non-specific DNA sites differ by only one order of magnitude, making it hard to understand how this protein recognizes its specific DNA targets in vivo. We describe here the structure of a p53 polypeptide containing both the DNA binding and oligomerization domains in complex with DNA. The structure reveals that sequence-specific DNA binding proceeds via an induced fit mechanism that involves a conformational switch in loop L1 of the p53 DNA binding domain. Analysis of loop L1 mutants demonstrated that the conformational switch allows DNA binding off-rates to be regulated independently of affinities. These results may explain the universal prevalence of conformational switching in sequence-specific DNA binding proteins and suggest that proteins like p53 rely more on differences in binding off-rates, than on differences in affinities, to recognize their specific DNA sites.

Growth phase and metal-dependent transcriptional regulation of the fecA genes in Helicobacter pylori.

Balancing metal uptake is essential for maintaining a proper intracellular metal concentration. Here, we report the transcriptional control exerted by the two metal-responsive regulators of Helicobacter pylori, Fur (iron-dependent ferric uptake regulator) and NikR (nickel-responsive regulator), on the three copies of the fecA genes present in this species. By monitoring the patterns of transcription throughout growth and in response to nickel, iron, and a metal chelator, we found that the expression of the three fecA genes is temporally regulated, responds to metals in different ways, and is selectively controlled by either one of the two regulators. fecA1 is expressed at a constant level throughout growth, and its expression is iron sensitive; the expression of fecA2 is mainly off, with minor expression coming up in late exponential phase. In contrast, the expression of fecA3 is maximal in early exponential phase, gradually decreases with time, and is repressed by nickel. The direct roles of Fur and NikR were studied both in vitro, by mapping the binding sites of each regulator on the promoter regions via DNase I footprinting analysis, and in vivo, by using primer extension analyses of the fecA transcripts in fur and nikR deletion strains. Overall, the results show that the expression of each fecA gene is finely tuned in response to metal availability, as well as during the bacterial growth phase, suggesting specific and dedicated functions for the three distinct FecA homologues.