Determinants of Replication-Fork Pausing at tRNA Genes in Saccharomyces cerevisiae.

Transfer RNA (tRNA) genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. 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 progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.

Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo.

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5' flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5' flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

Pif1-family helicases cooperatively suppress widespread replication-fork arrest at tRNA genes.

Saccharomyces cerevisiae expresses two Pif1-family helicases-Pif1 and Rrm3-which have been reported to play distinct roles in numerous nuclear processes. Here, we systematically characterized the roles of Pif1 helicases in replisome progression and lagging-strand synthesis in S. cerevisiae. We demonstrate that either Pif1 or Rrm3 redundantly stimulates strand displacement by DNA polymerase δ during lagging-strand synthesis. By analyzing replisome mobility in pif1 and rrm3 mutants, we show that Rrm3, with a partially redundant contribution from Pif1, suppresses widespread terminal arrest of the replisome at tRNA genes. Although both head-on and codirectional collisions induce replication-fork arrest at tRNA genes, head-on collisions arrest a higher proportion of replisomes. In agreement with this observation, we found that head-on collisions between tRNA transcription and replication are under-represented in the S. cerevisiae genome. We demonstrate that tRNA-mediated arrest is R-loop independent and propose that replisome arrest and DNA damage are mechanistically separable.

Verification of the Combimatrix influenza detection assay for the detection of influenza A subtype during the 2007-2008 influenza season in Toronto, Canada.

The increase in adamantine resistance in influenza A (H3N2) and the emergence of oseltamivir resistance in influenza A (H1N1) has necessitated the use of rapid methodologies to detect influenza subtype. The purpose of this study was to evaluate the CombiMatrix influenza detection system compared to the FDA approved Luminex Respiratory virus panel (RVP) assay for influenza A subtyping. Verification of the CombiMatrix influenza detection system was carried out using the Luminex RVP assay as a reference method. A limit of detection (LOD) series was performed using the Luminex and CombiMatrix systems with both influenza A H3N2 and H1N1 viruses. Seventy-five clinical specimens were used in the study. Of these, 16 were influenza A (H3N2) positive and five were influenza A (H1N1) positive. Fifty-four specimens were influenza A negative or "no call" (inconclusive) or could not be subtyped. The LOD of the Luminex RVP assay was found to be 0.3 TCID50s/mL for influenza A (H3N2) and 16 TCID50s/mL for influenza A (H1N1). The LOD of the CombiMatrix influenza detection system was 200 TCID50s/mL for influenza A (H3N2) and 16 000 TCID50s/mL for influenza A (H1N1). The sensitivity of the CombiMatrix influenza detection system was 95.2% and the specificity was 100%. The CombiMatrix influenza detection system is an effective methodology for influenza A subtype analysis, specifically in laboratories with a constrained budget or limited molecular capabilities.

Characterization of culture-positive adenovirus serotypes from respiratory specimens in Toronto, Ontario, Canada: September 2007-June 2008.

This study describes the prevalence of culture-positive adenovirus serotypes in culture-positive respiratory specimens sent to the Central Public Health Laboratory, Toronto, Ontario, Canada for the period September 2007-June 2008. Total nucleic acid was extracted from virus cultures using an automated extraction method followed by polymerase chain reaction and Sanger sequencing of the adenovirus hexon gene hypervariable region 7. 73% of specimens (n = 70) were from patients < or = 4 years of age. Of the 96 adenovirus isolates, the most common identified serotypes were serotype 3 (n = 44, 46%), serotype 2 (n = 25, 26%), serotype 1 (n = 17, 18%), and serotype 21 (n = 5, 5%). Adenovirus serotype 14 was not found in this study group. The leading serotype, Ad3, was identified throughout the duration of the study period. Molecular methods allow for the determination of circulating adenovirus serotypes and be used to document the spread of highly virulent adenoviral serotypes into a region.