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The THP1-SAC3-SUS1-CDC31 Complex Works in Transcription Elongation-mRNA Export Preventing RNA-mediated Genome Instability

The eukaryotic THO/TREX complex, involved in mRNP biogenesis, plays a key role in the maintenance of genome integrity in yeast. mRNA export factors such as Thp1-Sac3 also affect genome integrity, but their mutations have other phenotypes different from those of THO/TREX. Sus1 is a novel component of SAGA transcription factor that also associates with Thp1-Sac3, but little is known about its effect on genome instability and transcription. Here we show that Thp1, Sac3, and Sus1 form a functional unit with a role in mRNP biogenesis and maintenance of genome integrity that is independent of SAGA. Importantly, the effects of ribozyme-containing transcription units, RNase H, and the action of human activation-induced cytidine deaminase on transcription and genome instability are consistent with the possibility that R-loops are formed in Thp1-Sac3-Sus1-Cdc31 as in THO mutants. Our data reveal that Thp1-Sac3-Sus1-Cdc31, together with THO/TREX, define a specific pathway connecting transcription elongation with export via an RNA-dependent dynamic process that provides a feedback mechanism for the control of transcription and the preservation of genetic integrity of transcribed DNA regions.

Genome-wide analysis of factors affecting transcription elongation and DNA repair: a new role for PAF and Ccr4-not in transcription-coupled repair

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation.

The Rad50 coiled-coil domain is indispensable for Mre11 complex functions.

The Mre11 complex (Mre11, Rad50 and Xrs2 in Saccharomyces cerevisiae) influences diverse functions in the DNA damage response. The complex comprises the globular DNA-binding domain and the Rad50 hook domain, which are linked by a long and extended Rad50 coiled-coil domain. In this study, we constructed rad50 alleles encoding truncations of the coiled-coil domain to determine which Mre11 complex functions required the full length of the coils. These mutations abolished telomere maintenance and meiotic double-strand break (DSB) formation, and severely impaired homologous recombination, indicating a requirement for long-range action. Nonhomologous end joining, which is probably mediated by the globular domain of the Mre11 complex, was also severely impaired by alteration of the coiled-coil and hook domains, providing the first evidence of their influence on this process. These data show that functions of Mre11 complex are integrated by the coiled coils of Rad50.

Architecture and nucleic acids recognition mechanism of the THO complex, an mRNP assembly factor

The THO complex is a key factor in co‐transcriptional formation of export‐competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three‐dimensional structures of five‐subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C‐terminus using various labelling techniques. In the case of Tex1, a β‐propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C‐terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.

Distinct Roles of Mus81, Yen1, Slx1-Slx4, and Rad1 Nucleases in the Repair of Replication-Born Double-Strand Breaks by Sister Chromatid Exchange

ABSTRACT Most spontaneous DNA double-strand breaks (DSBs) arise during replication and are repaired by homologous recombination (HR) with the sister chromatid. Many proteins participate in HR, but it is often difficult to determine their in vivo functions due to the existence of alternative pathways. Here we take advantage of an in vivo assay to assess repair of a specific replication-born DSB by sister chromatid recombination (SCR). We analyzed the functional relevance of four structure-selective endonucleases (SSEs), Yen1, Mus81-Mms4, Slx1-Slx4, and Rad1, on SCR in Saccharomyces cerevisiae. Physical and genetic analyses showed that ablation of any of these SSEs leads to a specific SCR decrease that is not observed in general HR. Our work suggests that Yen1, Mus81-Mms4, Slx4, and Rad1, but not Slx1, function independently in the cleavage of intercrossed DNA structures to reconstitute broken replication forks via HR with the sister chromatid. These unique effects, which have not been detected in other studies unless double mutant combinations were used, indicate the formation of distinct alternatives for the repair of replication-born DSBs that require specific SSEs.

A novel assay identifies transcript elongation roles for the Nup84 complex and RNA processing factors

To clarify the role of a number of mRNA processing factors in transcription elongation, we developed an in vivo assay for direct analysis of elongation on chromatin. The assay relies on two substrates containing two G‐less cassettes separated by either a long and GC‐rich or a short and GC‐poor DNA sequence (G‐less‐based run‐on (GLRO) assay). We demonstrate that PAF, THSC/TREX‐2, SAGA, the exosome component Rrp6 and two subunits of cleavage factor IA (Rna14 and Rna15) are required for efficient transcription elongation, in contrast to some results obtained using other assays. Next, we undertook a mutant screen and found out that the Nup84 nucleoporin complex is also required for transcription elongation, as confirmed by the GLRO assay and RNA polymerase II chromatin immunoprecipitations. Therefore, in addition to showing that the GLRO assay is a sensitive and reliable method for the analysis of elongation in vivo, this study provides evidence for a new role of the Nup84 complex and a number of mRNA processing factors in transcription elongation that supports a connection of pre‐mRNA processing and nuclear export with transcription elongation.

Different physiological relevance of yeast THO/TREX subunits in gene expression and genome integrity

THO/TREX is a conserved nuclear complex that functions in mRNP biogenesis and plays a role in preventing the transcription-associated genetic instability. THO is composed of Tho2, Hpr1, Mft1 and Thp2 subunits, which associate with the Sub2-Yra1 export factors and Tex1 to form the TREX complex. To compare the functional relevance of the different THO/TREX subunits, we determined the effect of their null mutations on mRNA accumulation and recombination. Unexpectedly, we noticed that a full deletion of HPR1, hpr1ΔK, conferred stronger hyper-recombination phenotype and gene expression defects than did hpr1ΔH, the allele encoding a C-terminal truncated protein which was used in most previous studies. We show that tho2Δ and, to a lesser extent, hpr1ΔK are the THO mutations with the highest impact on all phenotypes, and that sub2Δ shows a similar transcription-dependent hyper-recombination phenotype and in vivo transcription impairment as hpr1ΔK and tho2Δ. Recombination and transcription analyses indicate that THO/TREX mutants share a moderate but significant effect on gene conversion and ectopic recombination, as well as transcription impairment of even short and low GC-content genes. Our data provide new information on the relevance of these proteins in mRNP biogenesis and in the maintenance of genomic integrity.

Nab2 functions in the metabolism of RNA driven by polymerases II and III

The hnRNP Nab2 is associated with actively transcribed RNAPII and RNAPIII genes. Nab2 has a function in RNAPII transcription and participates in tRNA metabolism and ribosomal subunit export, and, as a consequence, nab2 mutations confer translation defects. Results support Nab2 as a key regulator of gene expression.

Different genetic requirements for repair of replication-born double-strand breaks by sister-chromatid recombination and break-induced replication

Homologous recombination (HR) is the major mechanism used to repair double-strand breaks (DSBs) that result from replication, but a study of repair of DSBs specifically induced during S-phase is lacking. Using an inverted-repeat assay in which a DSB is generated by the encountering of the replication fork with nicks, we can physically detect repair by sister-chromatid recombination (SCR) and intra-chromatid break-induced replication (IC-BIR). As expected, both events depend on Rad52, but, in contrast to previous data, both require Rad59, suggesting a prominent role of Rad59 in repair of replication-born DSBs. In the absence of Rad51, SCR is severely affected while IC-BIR increases, a phenotype that is also observed in the absence of Rad54 but not of its paralog Rdh54/Tid1. These data are consistent with SCR occurring by Rad51-dependent mechanisms assisted by Rad54, and indicate that in the absence of strand exchange-dependent SCR, breaks can be channeled to IC-BIR, which works efficiently in the absence of Rad51. Our study provides molecular evidence for inversions between repeats occurring by BIR followed by single-strand annealing (SSA) in the absence of strand exchange.

Interdependence of the Rad50 Hook and Globular Domain Functions

Rad50 contains a conserved Zn(2+) coordination domain (the Rad50 hook) that functions as a homodimerization interface. Hook ablation phenocopies Rad50 deficiency in all respects. Here, we focused on rad50 mutations flanking the Zn(2+)-coordinating hook cysteines. These mutants impaired hook-mediated dimerization, but recombination between sister chromatids was largely unaffected. This may reflect that cohesin-mediated sister chromatid interactions are sufficient for double-strand break repair. However, Mre11 complex functions specified by the globular domain, including Tel1 (ATM) activation, nonhomologous end joining, and DNA double-strand break end resection were affected, suggesting that dimerization exerts a broad influence on Mre11 complex function. These phenotypes were suppressed by mutations within the coiled-coil and globular ATPase domains, suggesting a model in which conformational changes in the hook and globular domains are transmitted via the extended coils of Rad50. We propose that transmission of spatial information in this manner underlies the regulation of Mre11 complex functions.