Replication-fork arrest at tRNA genes in S. cerevisiae does not require tRNA transcription and is facilitated by topoisomerases and Rad18-dependent repair pathways

tRNA genes (tDNAs) are widely studied sites of replication-fork arrest and genome instability in the budding yeast Saccharomyces cerevisiae. tDNAs are extremely highly transcribed, and serve as constitutive condensin binding sites. Although tRNA transcription by RNA polymerase III (RNAPIII) has previously been identified as stimulating replication-fork arrest at these loci, the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of transcription factor binding, transcription, topoisomerase activity, and condensin-mediated clustering to replication-fork arrest at tDNAs in yeast. We show that a polar block to replication is maintained at tDNAs even when tRNA transcription is abolished by depletion of one or more subunits of RNAPIII. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication in the same background. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork mobility past tDNAs is unaffected by the global depletion of condensin from the nucleus, but can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways. Overall design: Factors with the potential to block replication forks at tDNAs were depleted. The resultant changes in fork progression were quantified as the ratio of Watson:Crick-mapping Okazaki fragments from high-throughput sequencing data.