Article

Highly Transcribed RNA Polymerase II Genes Are Impediments to Replication Fork Progression in Saccharomyces cerevisiae

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA.
Molecular cell (Impact Factor: 14.02). 07/2009; 34(6):722-34. DOI: 10.1016/j.molcel.2009.05.022
Source: PubMed

ABSTRACT

Replication forks face multiple obstacles that slow their progression. By two-dimensional gel analysis, yeast forks pause at stable DNA protein complexes, and this pausing is greatly increased in the absence of the Rrm3 helicase. We used a genome-wide approach to identify 96 sites of very high DNA polymerase binding in wild-type cells. Most of these binding sites were not previously identified pause sites. Rather, the most highly represented genomic category among high DNA polymerase binding sites was the open reading frames (ORFs) of highly transcribed RNA polymerase II genes. Twice as many pause sites were identified in rrm3 compared with wild-type cells, as pausing in this strain occurred at both highly transcribed RNA polymerase II genes and the previously identified protein DNA complexes. ORFs of highly transcribed RNA polymerase II genes are a class of natural pause sites that are not exacerbated in rrm3 cells.

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    • "Interestingly, INO80C can down-regulate transcription by repressing short-lived noncoding RNA at intergenic sites (Alcid and Tsukiyama 2014), possibly by restricting accessibility for RNA polymerase (Xue et al. 2015). In S-phase cells, transcription and replication compete for the same DNA template, making the transcriptional machinery a frequently encountered obstacle for replication forks (Gonzalez-Aguilera et al. 2008;Azvolinsky et al. 2009). Several mechanisms minimize the negative impact of transcription on DNA replication independently of the checkpoint. "
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    ABSTRACT: Little is known about how cells ensure DNA replication in the face of RNA polymerase II (RNAPII)-mediated transcription, especially under conditions of replicative stress. Here we present genetic and proteomic analyses from budding yeast that uncover links between the DNA replication checkpoint sensor Mec1-Ddc2 (ATR-ATRIP), the chromatin remodeling complex INO80C (INO80 complex), and the transcription complex PAF1C (PAF1 complex). We found that a subset of chromatin-bound RNAPII is degraded in a manner dependent on Mec1, INO80, and PAF1 complexes in cells exposed to hydroxyurea (HU). On HU, Mec1 triggers the efficient removal of PAF1C and RNAPII from transcribed genes near early firing origins. Failure to evict RNAPII correlates inversely with recovery from replication stress: paf1Δ cells, like ino80 and mec1 mutants, fail to restart forks efficiently after stalling. Our data reveal unexpected synergies between INO80C, Mec1, and PAF1C in the maintenance of genome integrity and suggest a mechanism of RNAPII degradation that reduces transcription-replication fork collision.
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    • "Transcription-associated recombination (TAR) is generally strongest when the transcription machinery and a replication fork approach each other head-on (Prado and Aguilera 2005), but there is also an orientation-independent component to such conflicts (Azvolinsky et al. 2009). "
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    ABSTRACT: Two types of RNA:DNA associations can lead to genome instability: the formation of R-loops during transcription and the incorporation of ribonucleotide monophosphates (rNMPs) into DNA during replication. Both ribonuclease (RNase) H1 and RNase H2 degrade the RNA component of R-loops, whereas only RNase H2 can remove one or a few rNMPs from DNA. We performed high-resolution mapping of mitotic recombination events throughout the yeast genome in diploid strains of Saccharomyces cerevisiae lacking RNase H1 (rnh1Δ), RNase H2 (rnh201Δ), or both RNase H1 and RNase H2 (rnh1Δ rnh201Δ). We found little effect on recombination in the rnh1Δ strain, but elevated recombination in both the rnh201Δ and the double-mutant strains; levels of recombination in the double mutant were about 50% higher than in the rnh201 single-mutant strain. An rnh201Δ mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase ε (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Polε. This result suggests that the elevated recombination observed in the absence of only RNase H2 is primarily a consequence of R loops rather than misincorporated rNMPs.
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