Impairment of replication fork progression mediates RNA polII transcription-associated recombination

Abstract

Homologous recombination safeguards genome integrity, but it can also cause genome instability of important consequences for cell proliferation and organism development. Transcription induces recombination, as shown in prokaryotes and eukaryotes for both spontaneous and developmentally regulated events such as those responsible for immunoglobulin class switching. Deciphering the molecular basis of transcription-associated recombination (TAR) is important in understanding genome instability. Using novel plasmid-borne recombination constructs in Saccharomyces cerevisiae, we show that RNA polymerase II (RNAPII) transcription induces recombination by impairing replication fork progression. RNAPII transcription concomitant to head-on oncoming replication causes a replication fork pause (RFP) that is linked to a significant increase in recombination. However, transcription that is codirectional with replication has little effect on replication fork progression and recombination. Transcription occurring in the absence of replication does not affect either recombination or replication fork progression. The Rrm3 helicase, which is required for replication fork progression through nucleoprotein complexes, facilitates replication through the transcription-dependent RFP site and reduces recombination. Therefore, our work provides evidence that one mechanism responsible for TAR is RNAP-mediated replication impairment.

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

“…This effect has been attributed to an enhanced replication block when the transcription machinery is directed toward an oncoming replication fork. Such transcription-dependent replication pause/stall sites have been observed in yeast [17,18] as well as in bacteria [19]. In addition, in E. coli, it has been observed that the manner in which a given DNA lesion is dealt with depends on the direction of replication [20].…”

“…A recent report has demonstrated that the relative orientation of the replication fork with respect to the direction of transcription indeed plays a significant role in transcription-associated recombination in a plasmid-based assay system [17]. This effect has been attributed to an enhanced replication block when the transcription machinery is directed toward an oncoming replication fork.…”

Activation-induced cytidine deaminase action is strongly stimulated by mutations of the THO complex

“…Although recombination is also enhanced by transcription, the mechanisms underlying transcription-associated recombination may be different from those of TAM. Thus, impairment of replication progression by transcription may be a critical factor triggering transcriptionassociated recombination (43,44), but this is not obvious for TAM. Our study reveals that THO mutations lead to a low hypermutator phenotype in the LAUR system, which is based on a highly expressed lacZ gene (Fig.…”

Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1

“…The facts that replisome stalling is not normal when RNA polymerase II collides head-to-head with the replisome in the genome and that eukaryotic RNA polymerase II-associated TAR has been reported only for plasmidbased transcription support the existence of such mechanisms (17)(18)(19). In budding yeast, Mec1 checkpoint kinase activity has been shown to be required to prevent so-called ''replication slow zones'' from becoming unstable (20).…”

“…Transcription-associated recombination (TAR) initiated via collisions of RNA polymerase with the replisome has also been suggested as a potential source of instability for genes transcribed by RNA polymerase II (16,17); however, such encounters between RNA polymerases and the replisome are relatively common occurrences within S-phase of the cell division cycle, so highly effective mechanisms must exist to prevent the generation of recombinogenic lesions. The facts that replisome stalling is not normal when RNA polymerase II collides head-to-head with the replisome in the genome and that eukaryotic RNA polymerase II-associated TAR has been reported only for plasmidbased transcription support the existence of such mechanisms (17)(18)(19).…”