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.

Hpr1 forms, together with Tho2, Mft1, and Thp2, the THO complex, which controls transcription elongation and genome stability in Saccharomyces cerevisiae. Mutations in genes encoding the THO complex confer strong transcription-impairment and hyperrecombination phenotypes in the bacterial lacZ gene. In this work we demonstrate that Hpr1 is a factor required for transcription of long as well as G؉C-rich DNA sequences. Using different lacZ segments fused to the GAL1 promoter, we show that the negative effect of lacZ sequences on transcription depends on their distance from the promoter. In parallel, we show that transcription of either a long LYS2 fragment or the S. cerevisiae YAT1 G؉C-rich open reading frame fused to the GAL1 promoter is severely impaired in hpr1 mutants, whereas transcription of LAC4, the Kluyveromyces lactis ortholog of lacZ but with a lower G؉C content, is only slightly affected. The hyperrecombination behavior of the DNA sequences studied is consistent with the transcriptional defects observed in hpr1 cells. These results indicate that both length and G؉C content are important elements influencing transcription in vivo. We discuss their relevance for the understanding of the functional role of Hpr1 and, by extension, the THO complex.The control of genome stability is essential to ensure maintenance of genetic information in all cells of a living organism. Dysfunction of this control causes mutations and chromosomal aberrations that can give rise to loss of gene function, cell death, or irreversible changes in the cell program.Genetic recombination is required for mitotic DNA repair and for proper meiotic chromosome segregation. In addition, it may also be responsible for processes of genetic instability. A number of animal diseases, including cancer, originate by events of mitotic recombination between repeats that lead to chromosomal aberrations (34). Several elements have been described to enhance mitotic recombination, including DNA damage, replication defects, alteration of chromatin structure, and transcriptional activity (reviewed in reference 3). Ikeda and Matsumoto (26) first described the influence of transcription on recombination showing that recombination of phage was stimulated by transcription. In yeast, the first example of transcription-associated recombination was the finding that a hotspot of ribosomal DNA (rDNA) recombination, HOT1, was dependent on RNA polymerase I-driven transcription (55, 60). Thomas and Rothstein (56) extended transcription-induced recombination to sequences transcribed by RNA polymerase II (RNAPII). Additional examples of RNAPII-dependent recombination have been subsequently described in yeast (21,36,50) and mammalian cells (37, 57). Special mention must be made of the modulation of recombination at the immunoglobulin loci, as both V(D)J recombination (7, 31, 38) and class switching (15) are positively controlled by transcription.A gene linking transcription and genome instability in Saccharomyces cerevisae is HPR1, as hpr1 mutants show both increased l...

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...

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non-repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non-DSB DNA lesions, presumably single-stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle-regulated replicative and repair functions.