Loss of Topoisomerase I leads to R-loop-mediated transcriptional blocks during ribosomal RNA synthesis.
Genes Dev
; 24(14): 1546-58, 2010 Jul 15.
Article
em En
| MEDLINE
| ID: mdl-20634320
ABSTRACT
Pre-rRNA transcription by RNA Polymerase I (Pol I) is very robust on active rDNA repeats. Loss of yeast Topoisomerase I (Top1) generated truncated pre-rRNA fragments, which were stabilized in strains lacking TRAMP (Trf4/Trf5-Air1/Air2-Mtr4 polyadenylation complexes) or exosome degradation activities. Loss of both Top1 and Top2 blocked pre-rRNA synthesis, with pre-rRNAs truncated predominately in the 18S 5' region. Positive supercoils in front of Pol I are predicted to slow elongation, while rDNA opening in its wake might cause R-loop formation. Chromatin immunoprecipitation analysis showed substantial levels of RNA/DNA hybrids in the wild type, particularly over the 18S 5' region. The absence of RNase H1 and H2 in cells depleted of Top1 increased the accumulation of RNA/DNA hybrids and reduced pre-rRNA truncation and pre-rRNA synthesis. Hybrid accumulation over the rDNA was greatly exacerbated when Top1, Top2, and RNase H were all absent. Electron microscopy (EM) analysis revealed Pol I pileups in the wild type, particularly over the 18S. Pileups were longer and more frequent in the absence of Top1, and their frequency was exacerbated when RNase H activity was also lacking. We conclude that the loss of Top1 enhances inherent R-loop formation, particularly over the 5' region of the rDNA, imposing persistent transcription blocks when RNase H is limiting.
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Base de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Transcrição Gênica
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Precursores de RNA
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RNA Ribossômico 18S
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DNA Topoisomerases Tipo I
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article