The deleterious effects of transcription on genome integrity have been suggested by various observations (Aguilera, 2002
). Whenever the integrated process of transcript processing, packaging, and export in eukaryotes is disrupted, genome instability can be observed (Baaklini et al., 2004; Broccoli et al., 2004; Jimeno et al., 2002; Luna et al., 2005
). This has been shown to derive from R loops, which preferentially form when mRNP biogenesis is disrupted. Our data show that Sen1 helicase plays a pivotal role in the prevention of genome instability by recombination. A large fraction of this instability is transcription dependent and linked to the formation of R loop structures. The exact nature of these structures remains to be established, but we show their accumulation can still occur with normal mRNP biogenesis. If these structures are not removed by either Sen1 helicase or RNase H directed degradation, they can exert a deleterious effect on genome stability, as is further illustrated by SEN1
genetic interaction with HR genes. The occurrence of Rad52 foci, as a marker for ongoing recombination, shows that recombination is related to transcription, as well as to the presence of a functional Sen1 helicase domain. In summary, we suggest that R loop formation is more frequent than hitherto anticipated and requires active removal by helicases such as Sen1.
We suggest that as soon as the nascent transcript emerges from the polymerase body, mRNA packaging and R loop formation occur in kinetic competition (). A fragile equilibrium between protective mRNA packaging and the hiding of specific recognition sequences is likely to exist (Bucheli and Buratowski, 2005; Bucheli et al., 2007
). Therefore, RNA packaging is likely to be incomplete so leaving some transcript available for R loop formation. For pervasive CUT transcription, termination depends on NRD and by inference on Sen1 (Arigo et al., 2006b
). If R loops formed in sen1-1
extend to many CUT loci, then their accumulation, even if transient, would cover substantial regions of the genome. In both CUTs and mRNA coding genes, R loops could interfere with DNA replication, induce ssDNA breaks, or be recognized as recombination intermediates. Any of these possibilities could explain the essential need for DSB sensing proteins in sen1-1
(). However, the different genetic interactions of sen1-1
with HR or S phase checkpoint genes suggest structural and functional differences of the replication/recombinogenic intermediates that are formed (Gómez-González et al., 2009
). Alternatively, these differences may hint at a transcription-independent role of Sen1 in DNA damage repair that is yet to be uncovered.
Cotranscriptional Functions of Sen1
How may R loop accumulation in sen1-1
be related to its transcription termination defect? R loops were originally hypothesized to slow down transcription elongation, thereby enhancing termination (Proudfoot, 1989
). This would give time for the Rat1 5′-3′ exonuclease “torpedo” to catch up with Pol II but would require R loop resolution by an enzymatic activity such as Sen1 prior to degradation. Based on observations made on THO mutants, R loops have been suggested to interfere with transcription elongation (Huertas and Aguilera, 2003; Mason and Struhl, 2005
). Employing sen1-1
, in which transcript processing is normal, we predict that reduced steady-state RNA accumulation is due to reduced transcript elongation. Furthermore, the data presented here support the view that R loops preferentially form in termination regions. Thus, we employed the LNA/LNAT recombination substrates, anticipating that even though the CYC1
pA would not elicit termination (Kawauchi et al., 2008
), it should serve as a 3′ processing signal (C), promoting disassembly of THO and consequent R loop formation (Kim et al., 2004a
). Compared to LNA, sen1-1
recombination levels increased 2-fold in LNAT. This demonstrates for sen1-1
in contrast to THO mutants, that RNA cleavage in the context of a pA is not sufficient to relieve recombination. To reiterate this point, Figure S2
shows that in a ribozyme containing substrate, recombination levels in sen1-1
are reduced similar to hpr1Δ
(Huertas and Aguilera, 2003
). As both ribozyme cleaved ends are unprotected they are likely to be degraded and so reduce R loop forming substrate. However, this appears not to be the case if RNA in sen1-1
cells is cleaved at a pA, possibly as RNA downstream to the polyA signal may be less packaged and so temporarily protected from degradation by R loop formation. Although these studies require a more detailed biochemical analysis, we predict from these initial results that R loops may play a role in transcriptional termination.
In summary the molecular and genetic effects of Sen1 inactivation presented here reveal that Sen1 acts to protect the heavily transcribed genome from R loop-mediated DNA damage. Of note, mutations in the helicase domain of the human SEN1
gene ortholog SETX
(encoding Senataxin) cause the neurodegenerative diseases, Ataxia with Oculomotor Apraxia Type II (AOAII), and juvenile amyotrophic lateral sclerosis (ALS4). Like sen1-1
mutants show defects in transcription, RNA processing, and DNA damage repair (Moreira et al., 2004; Suraweera et al., 2007, 2009
). It remains to be established whether the tendency of transcription to induce R loop formation is a general feature of all eukaryotic genomes. It is possible that a range of dedicated helicases act to resolve these potentially harmful structures.