Recombination between DNA repeats in yeast hpr1Delta cells is linked to transcription elongation

Abstract

The induction of recombination by transcription activation has been documented in prokaryotes and eukaryotes. Unwinding of the DNA duplex, disruption of chromatin structure or changes in local supercoiling associated with transcription can be indirectly responsible for the stimulation of recombination. Here we provide genetic and molecular evidence for a specific mechanism of stimulation of recombination by transcription. We show that the induction of deletions between repeats in hpr1Δ cells of Saccharomyces cerevisiae is linked to transcription elongation. Molecular analysis of different direct repeat constructs reveals that deletions induced by hpr1Δ are specific for repeat constructs in which transcription initiating at an external promoter traverses particular regions of the DNA flanked by the repeats. Transcription becomes HPR1 dependent when elongating through such regions. Both the induction of deletions and the HPR1 dependence of transcription were abolished when a strong terminator was used to prevent transcription from proceeding through the DNA region flanked by the repeats. In contrast to previously reported cases of transcription‐induced recombination, there was no correlation between high levels of transcripts and high levels of recombination. Our study provides evidence that direct repeat recombination can be induced by transcriptional elongation.

Relationship between G+C content, ORF‐length and mRNA concentration in Saccharomyces cerevisiae

“…This effect has been observed in hpr mutants which lack one of the components of THO, a heteromeric protein complex formed by Tho2, Hpr1, Mft1 and Thp2 (Prado et al, 1997;Chávez and Aguilera, 1997;Piruat and Aguilera, 1998;Chávez et al, 2000).…”

“…Cells were grown to mid-log phase (OD 600 = 0.6) in rich YEPD medium at 30 • C. Total RNA was extracted according to standard procedures (Prado et al, 1997), and poly(A) + RNA was purified from total RNA using the Oligotex-dt30 mRNA purification kit (TAKARA). Poly(A) + RNA was amplified, biotin-labelled and hybridized to oligonucleotide arrays (GeneChip S98 arrays, Affimetrix, Santa Clara, CA).…”

Transcription-associated mutagenesis in yeast is directly proportional to the level of gene expression and influenced by the direction of DNA replication

“…Evidence for a direct role of transcription in stimulating recombination has come from the analysis of HPR1-deficient yeast strains, where up to 1000-fold increases in recombination between direct repeats have been observed [9]. This hyper-recombination phenotype in HPR1-deficient strains has been shown to be directly related to the movement of RNA polymerase II (RNAP) complexes through the recombination substrate; when transcription elongation was inhibited by the insertion of a transcription termination sequence between the two direct repeats, the hyper-recombination phenotype was abolished [10]. Subsequently, Hpr1 was shown to be a component of the THO/TREX complex, inactivation of which leads to the elongation and stabilization of RNA:DNA hybrids (R-loops) formed during transcription [11].…”

“…Also, at least for TAR, there is evidence that chromatin remodeling associated with the activation of transcription may be a contributing factor [14]. Finally, the stress resulting from transcription-associated changes in DNA topology/superhelicity may lead to nicks or breaks in DNA [10,15,16].…”

Advances in AAV-mediated gene transfer for the treatment of inherited disorders

“…On the other hand, it has been published that certain recombination events require transcription, and recombination might be involved in rAAV2 integration. 30,31 Despite existing limitations and concerns that still need to be addressed and resolved, gene transfer vectors based on recombinant AAVs still represent one of the most promising gene therapy systems and gain increasing popularity. Thus, it comes at no surprise that the first gene therapy applications based on rAAV vectors have entered the clinical stage.…”