Replication Fork Progression Is Impaired by Transcription in Hyperrecombinant Yeast Cells Lacking a Functional THO Complex

Abstract

THO/TREX is a conserved, eukaryotic protein complex operating at the interface between transcription and messenger ribonucleoprotein (mRNP) metabolism. THO mutations impair transcription and lead to increased transcription-associated recombination (TAR). These phenotypes are dependent on the nascent mRNA; however, the molecular mechanism by which impaired mRNP biogenesis triggers recombination in THO/TREX mutants is unknown. In this study, we provide evidence that deficient mRNP biogenesis causes slowdown or pausing of the replication fork in hpr1⌬ mutants. Impaired replication appears to depend on sequence-specific features since it was observed upon activation of lacZ but not leu2 transcription. Replication fork progression could be partially restored by hammerhead ribozyme-guided self-cleavage of the nascent mRNA. Additionally, hpr1⌬ increased the number of S-phase but not G 2 -dependent TAR events as well as the number of budded cells containing Rad52 repair foci. Our results link transcription-dependent genomic instability in THO mutants with impaired replication fork progression, suggesting a molecular basis for a connection between inefficient mRNP biogenesis and genetic instability.Genetic instability of a DNA fragment can be induced by transcription, a phenomenon referred to as transcription-associated recombination (TAR). Recombination has been shown to increase in actively transcribed genes in bacteria, yeasts, and humans (1). This is the case for RNA polymerase II (RNAPII)-dependent transcription, as first shown for yeast (43). Despite the ubiquity and relevance of TAR, its mechanism(s) remains unknown. Transcription-dependent hyperrecombination is a hallmark phenotype of mutants of the THO complex in the yeast S. cerevisiae (4,36). THO is a conserved, eukaryotic multiprotein complex, containing Hpr1, Mft1, Tho2, and Thp2 in yeast (5). Moreover, THO acts at the interface between transcription and mRNP export via its interaction with Sub2 and Yra1 in a highmolecular-weight complex termed TREX (19,40). THO/TREX components are recruited to an active gene during transcription elongation. Hpr1 directly interacts with Sub2 and facilitates the binding of Sub2 and Yra1 to nascent transcripts (46). Mutations in most factors involved in messenger RNP (mRNP) biogenesis and export, including Sub2, Yra1, Thp1-Sac3, Nab2, Mex67, and Mtr2, confer a gene expression defect and a transcription-dependent hyperrecombination phenotype comparable to that described for THO mutant strains (10, 19). The similarity of these phenotypes suggests that correct processing of a number of nuclear steps, leading to export-competent mRNP particles, is important in preventing transcription-dependent genomic instability (29). It remains to be seen whether TAR events stimulated in THO mutants result from the same mechanism(s) as spontaneous TAR events occurring in wild-type cells.DNA replication occurs during S phase of the cell cycle and is initiated at multiple origins of replication (20). Once established, a single replication fork wi...

An hpr1 Point Mutation That Impairs Transcription and mRNP Biogenesis without Increasing Recombination

“…We have recently shown that transcription impairment of hpr1⌬ along a highly transcribed lacZ gene causes a retardation of replication, observed as a slowdown of fork progression (43). This slowdown of fork progression could partially be suppressed by ribozyme-mediated cleavage of the mRNA.…”

“…All data were normalized with respect to the endogenous U3 signal. (43). While S-phase-specific transcription was capable of inducing recombination, G 2 -specific transcription was not.…”

Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins

“…TREX subunits have been shown to be important for maintenance of the genomic stability of transcribed sequences (31). Recent work indicates that mutants in the TREX subunit called Hpr1 lead to disrupted transcription and mRNP biogenesis that inhibit the movement of replication forks through areas of the genome undergoing transcription during S phase (42). As Yra1 is a member of the TREX complex, it is possible that yra1 mutants could also lead to defective mRNP biogenesis that disrupts DNA replication.…”

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.…”