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


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.

Download full-text


Available from: Jason D Lieb,
1 Follower
38 Reads
  • Source
    • "Another source of replication stress arises from transcription— replication collisions (Azvolinsky et al. 2009). Such collisions seem to play a central role in the maintenance of Chromosoma pericentromeric heterochromatin in fission yeast. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The eukaryotic genome can be roughly divided into euchromatin and heterochromatin domains that are structurally and functionally distinct. Heterochromatin is characterized by its high compaction that impedes DNA transactions such as gene transcription, replication, or recombination. Beyond its role in regulating DNA accessibility, heterochromatin plays essential roles in nuclear architecture, chromosome segregation, and genome stability. The formation of heterochromatin involves special histone modifications and the recruitment and spreading of silencing complexes that impact the higher-order structures of chromatin; however, its molecular nature varies between different chromosomal regions and between species. Although heterochromatin has been extensively characterized, its formation and maintenance throughout the cell cycle are not yet fully understood. The biggest challenge for the faithful transmission of chromatin domains is the destabilization of chromatin structures followed by their reassembly on a novel DNA template during genomic replication. This destabilizing event also provides a window of opportunity for the de novo establishment of heterochromatin. In recent years, it has become clear that different types of obstacles such as tight protein-DNA complexes, highly transcribed genes, and secondary DNA structures could impede the normal progression of the replisome and thus have the potential to endanger the integrity of the genome. Multiple studies carried out in different model organisms have demonstrated the capacity of such replisome impediments to favor the formation of heterochromatin. Our review summarizes these reports and discusses the potential role of replication stress in the formation and maintenance of heterochromatin and the role that silencing proteins could play at sites where the integrity of the genome is compromised.
    Chromosoma 10/2015; DOI:10.1007/s00412-015-0545-6 · 4.60 Impact Factor
  • Source
    • "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). "
    [Show abstract] [Hide abstract]
    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.
    Genetics 09/2015; DOI:10.1534/genetics.115.182725 · 5.96 Impact Factor
  • Source
    • "s and Kadonaga , 2014 ) . For yeast Pif1 and Sen1 DNA / RNA helicases a role in preventing replication – transcription interference has been also ascertained in vivo . In particular , members of the Pif1 helicase family assist fork pro - gression through several type of natural barriers , including tran - scription blocks ( Ivessa et al . , 2003 ; Azvolinsky et al . , 2009 ; Paeschke et al . , 2011 ) , while Sen1 is specifically required to prevent RNA : DNA hybrids accumulation at the fork in head - on encounters with RNAPII transcribed genes ( Alzu et al . , 2012 ) . In human , both Senataxin / SETX , the ortholog of Sen1 , and Aquarius , an helicase structurally related to Senataxin , prevent DNA damag"
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA replication and transcription are vital cellular processes during which the genetic information is copied into complementary DNA and RNA molecules. Highly complex machineries required for DNA and RNA synthesis compete for the same DNA template, therefore being on a collision course. Unscheduled replication-transcription clashes alter the gene transcription program and generate replication stress, reducing fork speed. Molecular pathways and mechanisms that minimize the conflict between replication and transcription have been extensively characterized in prokaryotic cells and recently identified also in eukaryotes. A pathological outcome of replication-transcription collisions is the formation of stable RNA:DNA hybrids in molecular structures called R-loops. Growing evidence suggests that R-loop accumulation promotes both genetic and epigenetic instability, thus severely affecting genome functionality. In the present review, we summarize the current knowledge related to replication and transcription conflicts in eukaryotes, their consequences on genome instability and the pathways involved in their resolution. These findings are relevant to clarify the molecular basis of cancer and neurodegenerative diseases.
    Frontiers in Genetics 04/2015; 6. DOI:10.3389/fgene.2015.00166
Show more