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A distinct role for recombination repair factors in an early cellular response to transcription-replication conflicts.

ABSTRACT: Transcription-replication (T-R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T-R conflicts, our understanding of the genetic and temporal nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T-R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-independent function of FANCD2. A hallmark of the TRe response is the rapid co-localization of these three DNA repair factors at sites of T-R collisions. We find that the TRe response relies on basal activity of the ATR kinase, yet it does not lead to hyperactivation of this key checkpoint protein. Furthermore, specific abrogation of the TRe response leads to DNA damage in mitosis, and promotes chromosome instability and cell death. Collectively our findings identify a new role for these well-established tumor suppressor proteins at an early stage of the cellular response to conflicts between DNA transcription and replication.

SUBMITTER: Shao X

PROVIDER: S-EPMC7261159 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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A distinct role for recombination repair factors in an early cellular response to transcription-replication conflicts.

Shao Xin X Joergensen Amalie M AM Howlett Niall G NG Lisby Michael M Oestergaard Vibe H VH

Nucleic acids research 20200601 10

Transcription-replication (T-R) conflicts are profound threats to genome integrity. However, whilst much is known about the existence of T-R conflicts, our understanding of the genetic and temporal nature of how cells respond to them is poorly established. Here, we address this by characterizing the early cellular response to transient T-R conflicts (TRe). This response specifically requires the DNA recombination repair proteins BLM and BRCA2 as well as a non-canonical monoubiquitylation-indepen ...[more]

PMID: 32329774

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Project description:Several mechanisms that increase the rate of mutagenesis across the entire genome have been identified; however, how the rate of evolution might be promoted in individual genes is unclear. Most genes in bacteria are encoded on the leading strand of replication. This presumably avoids the potentially detrimental head-on collisions that occur between the replication and transcription machineries when genes are encoded on the lagging strand. Here we identify the ubiquitous (core) genes in Bacillus subtilis and determine that 17% of them are on the lagging strand. We find a higher rate of point mutations in the core genes on the lagging strand compared with those on the leading strand, with this difference being primarily in the amino-acid-changing (nonsynonymous) mutations. We determine that, overall, the genes under strong negative selection against amino-acid-changing mutations tend to be on the leading strand, co-oriented with replication. In contrast, on the basis of the rate of convergent mutations, genes under positive selection for amino-acid-changing mutations are more commonly found on the lagging strand, indicating faster adaptive evolution in many genes in the head-on orientation. Increased gene length and gene expression amounts are positively correlated with the rate of accumulation of nonsynonymous mutations in the head-on genes, suggesting that the conflict between replication and transcription could be a driving force behind these mutations. Indeed, using reversion assays, we show that the difference in the rate of mutagenesis of genes in the two orientations is transcription dependent. Altogether, our findings indicate that head-on replication-transcription conflicts are more mutagenic than co-directional conflicts and that these encounters can significantly increase adaptive structural variation in the coded proteins. We propose that bacteria, and potentially other organisms, promote faster evolution of specific genes through orientation-dependent encounters between DNA replication and transcription.

Project description:UnlabelledEfficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.ImportanceHead-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.

Dataset Information

A distinct role for recombination repair factors in an early cellular response to transcription-replication conflicts.

Publications

A distinct role for recombination repair factors in an early cellular response to transcription-replication conflicts.

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