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.

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C. elegans THSC/TREX-2 deficiency causes replication stress and genome instability

Journal of Cell Science ◽

10.1242/jcs.258435 ◽

2021 ◽

Author(s):

Angelina Zheleva ◽

Lola P Camino ◽

Nuria Fernández-Fernández ◽

María García-Rubio ◽

Peter Askjaer ◽

...

Keyword(s):

Dna Damage ◽

Damage Accumulation ◽

Genome Instability ◽

Underlying Mechanism ◽

Rnase H ◽

Genome Integrity ◽

Dna Damage Checkpoint ◽

C Elegans ◽

Mrnp Biogenesis

Transcription is an essential process of DNA metabolism, yet it makes DNA more susceptible to DNA damage. THSC/TREX-2 is a conserved eukaryotic protein complex with a key role in mRNP biogenesis and maturation that prevents genome instability. One source of such instability is linked to transcription as shown in yeast and human cells, but the underlying mechanism and whether is universal is still unclear. To get further insight in the putative role of THSC/TREX-2 in genome integrity we have used Caenorhabditis elegans mutants of the THP-1 and DSS-1 members of THSC/TREX-2. These mutants show similar defective meiosis, DNA damage accumulation and activation of the DNA damage checkpoint. However, they differ regarding replication defects as determined by dUTP incorporation in the germline. Interestingly, this specific thp-1 phenotype can be partially rescued by overexpression of RNase H. Furthermore, both mutants show a mild increase in the H3S10P mark previously shown to be linked to DNA-RNA hybrid-mediated genome instability. These data support the view that both THSC/TREX-2 factors prevent transcription-associated DNA damage derived from DNA-RNA hybrid accumulation by separate means.

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Single-molecule fluorescence studies on cotranscriptional G-quadruplex formation coupled with R-loop formation

Nucleic Acids Research ◽

10.1093/nar/gkaa695 ◽

2020 ◽

Vol 48 (16) ◽

pp. 9195-9203

Author(s):

Gunhyoung Lim ◽

Sungchul Hohng

Keyword(s):

Gene Regulation ◽

Single Molecule ◽

Genome Instability ◽

Feedback Mechanism ◽

Rnase H ◽

Formation Mechanisms ◽

Loop Formation ◽

Single Molecule Fluorescence ◽

Fluorescence Studies ◽

G Quadruplex

Abstract G-quadruplex (GQ) is formed at various regions of DNA, including telomeres of chromosomes and regulatory regions of oncogenes. Since GQ is important in both gene regulation and genome instability, the biological and medical implications of this abnormal DNA structure have been intensively studied. Its formation mechanisms, however, are not clearly understood yet. We report single-molecule fluorescence experiments to monitor the cotranscriptional GQ formation coupled with R-loop formation using T7 RNA polymerase. The GQ is formed very rarely per single-round transcription. R-loop formation precedes and facilitates GQ formation. Once formed, some GQs are extremely stable, resistant even to RNase H treatment, and accumulate in multiple-round transcription conditions. On the other hand, GQ existing in the non-template strand promotes the R-loop formation in the next rounds of transcription. Our study clearly shows the existence of a positive feedback mechanism of GQ and R-loop formations, which may possibly contribute to gene regulation and genome instability.

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Attracting AID to targets of somatic hypermutation

Journal of Experimental Medicine ◽

10.1084/jem.20090821 ◽

2010 ◽

Vol 207 (2) ◽

pp. 405-415 ◽

Cited By ~ 43

Author(s):

Atsushi Tanaka ◽

Hong Ming Shen ◽

Sarayu Ratnam ◽

Prashant Kodgire ◽

Ursula Storb

Keyword(s):

Target Gene ◽

Somatic Hypermutation ◽

Cytidine Deaminase ◽

The Other ◽

Genome Integrity ◽

Cis Elements ◽

Transcription Activity ◽

Nuclear Extracts ◽

Activation Induced Cytidine Deaminase ◽

Chromatin Acetylation

The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes requires activation-induced cytidine deaminase (AID). Although mistargeting of AID is detrimental to genome integrity, the mechanism and the cis-elements responsible for targeting of AID are largely unknown. We show that three CAGGTG cis-elements in the context of Ig enhancers are sufficient to target SHM to a nearby transcribed gene. The CAGGTG motif binds E47 in nuclear extracts of the mutating cells. Replacing CAGGTG with AAGGTG in the construct without any other E47 binding site eliminates SHM. The CA versus AA effect requires AID. CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity. The other cis-elements of Ig enhancers alone cannot attract the SHM machinery. Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context. Ig genes are the most highly mutated genes, presumably because of multiple CAGGTG motifs within the Ig genes, high transcription activity, and the presence of other cooperating elements in Ig enhancers.

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