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Transcription shapes DNA replication initiation and termination in human cells.

ABSTRACT: Although DNA replication is a fundamental aspect of biology, it is not known what determines where DNA replication starts and stops in the human genome. We directly identified and quantitatively compared sites of replication initiation and termination in untransformed human cells. We found that replication preferentially initiates at the transcription start site of genes occupied by high levels of RNA polymerase II, and terminates at their polyadenylation sites, thereby ensuring global co-directionality of transcription and replication, particularly at gene 5' ends. During replication stress, replication initiation is stimulated downstream of genes and termination is redistributed to gene bodies; this globally reorients replication relative to transcription around gene 3' ends. These data suggest that replication initiation and termination are coupled to transcription in human cells, and propose a model for the impact of replication stress on genome integrity.

SUBMITTER: Chen YH

PROVIDER: S-EPMC6320713 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Transcription shapes DNA replication initiation to preserve genome integrity.

Project description:BackgroundEarly DNA replication occurs within actively transcribed chromatin compartments in mammalian cells, raising the immediate question of how early DNA replication coordinates with transcription to avoid collisions and DNA damage.ResultsWe develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones in both mouse and human cells. The identified early replication initiation zones fall in open chromatin compartments and are mutually exclusive with transcription elongation. Of note, early replication initiation zones are mainly located in non-transcribed regions adjacent to transcribed regions. Mechanistically, we find that RNA polymerase II actively redistributes the chromatin-bound mini-chromosome maintenance complex (MCM), but not the origin recognition complex (ORC), to actively restrict early DNA replication initiation outside of transcribed regions. In support of this finding, we detect apparent MCM accumulation and DNA replication initiation in transcribed regions due to anchoring of nuclease-dead Cas9 at transcribed genes, which stalls RNA polymerase II. Finally, we find that the orchestration of early DNA replication initiation by transcription efficiently prevents gross DNA damage.ConclusionRNA polymerase II redistributes MCM complexes, but not the ORC, to prevent early DNA replication from initiating within transcribed regions. This RNA polymerase II-driven MCM redistribution spatially separates transcription and early DNA replication events and avoids the transcription-replication initiation collision, thereby providing a critical regulatory mechanism to preserve genome stability.

| S-EPMC8188667 | biostudies-literature

Transcription shapes DNA replication initiation to preserve genome integrity

Project description:We develop a high-throughput nucleoside analog incorporation sequencing assay and identify thousands of early replication initiation zones (ERIZs) in both mouse and human cells. The identified ERIZs fall in open chromatin compartments while are mutually exclusive with transcription elongation and occupy mainly non-transcribed regions adjacent to transcribed regions. Furthermore, we reveal that RNA polymerase II actively redistributes the chromatin-encircled mini-chromosome maintenance (MCM) complex but not the origin-recognition complex (ORC) to actively restrict early DNA replication initiation outside of transcribed regions. The coupling of RNA polymerase II and MCM is further validated by detected MCM accumulation and DNA replication initiation after RNA polymerase II stalling via anchoring nuclease-dead Cas9 at the transcribed genes. Importantly, we also find that the orchestration of DNA replication initiation by transcription can efficiently prevent gross DNA damage.

2021-05-20 | GSE174680 | GEO

The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA.

Project description:The mammalian mitochondrial transcription termination factor mTERF binds with high affinity to a site within the tRNA(Leu(UUR)) gene and regulates the amount of read through transcription from the ribosomal DNA into the remaining genes of the major coding strand of mitochondrial DNA (mtDNA). Electrophoretic mobility shift assays (EMSA) and SELEX, using mitochondrial protein extracts from cells induced to overexpress mTERF, revealed novel, weaker mTERF-binding sites, clustered in several regions of mtDNA, notably in the major non-coding region (NCR). Such binding in vivo was supported by mtDNA immunoprecipitation. Two-dimensional neutral agarose gel electrophoresis (2DNAGE) and 5' end mapping by ligation-mediated PCR (LM-PCR) identified the region of the canonical mTERF-binding site as a replication pause site. The strength of pausing was modulated by the expression level of mTERF. mTERF overexpression also affected replication pausing in other regions of the genome in which mTERF binding was found. These results indicate a role for TERF in mtDNA replication, in addition to its role in transcription. We suggest that mTERF could provide a system for coordinating the passage of replication and transcription complexes, analogous with replication pause-region binding proteins in other systems, whose main role is to safeguard the integrity of the genome whilst facilitating its efficient expression.

| S-EPMC2095818 | biostudies-literature

XRN2 Links Transcription Termination to DNA Damage and Replication Stress.

Project description:XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.

| S-EPMC4954731 | biostudies-literature

Transcription shapes DNA replication initiation to preserve genome integrity

Project description:Transcription shapes DNA replication initiation to preserve genome integrity

| PRJNA731108 | ENA

Detection of template strand switching during initiation and termination of DNA replication of porcine circovirus.

Project description:Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle "melting-pot" model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a "melted" configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a "gene correction" or "terminal repeat correction" mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.

| S-EPMC374294 | biostudies-literature

Phosphorylation of TFIIB links transcription initiation and termination.

Project description:The general transcription factor TFIIB plays a central role in preinitiation complex (PIC) assembly and the recruitment of RNA polymerase II (RNA pol II) to the promoter. Recent studies have revealed that TFIIB engages in contact with the transcription termination region and also with complexes that are involved in 3' end processing and/or termination. Here we report that TFIIB can be phosphorylated within the N terminus at serine 65 in vivo and that the phosphorylated form of TFIIB is present within (PICs). Surprisingly, TFIIB serine 65 phosphorylation is required after the phosphorylation of serine 5 of RNA pol II C-terminal domain (CTD) has occurred, but before productive transcription initiation begins. We show that phosphorylation of TFIIB at serine 65 regulates the interaction between TFIIB and the CstF-64 component of the CstF 3' cleavage and polyadenylation complex. This directs the recruitment of CstF (cleavage stimulatory factor) to the terminator and also the recruitment of the CstF and CPSF (cleavage and polyadenylation specific factor) complexes to the promoter. Our results reveal that phosphorylation of TFIIB is a critical event in transcription that links the gene promoter and terminator and triggers initiation by RNA pol II.

| S-EPMC2849011 | biostudies-literature

DNA replication initiation shapes the mutational landscape and expression of the human genome

Project description:This SuperSeries is composed of the SubSeries listed below.

2022-11-15 | GSE202799 | GEO

DNA replication origin interference increases the spacing between initiation events in human cells.

Project description:Mammalian DNA replication origins localize to sites that range from base pairs to tens of kilobases. A regular distribution of initiations in individual cell cycles suggests that only a limited number of these numerous potential start sites are converted into activated origins. Origin interference can silence redundant origins; however, it is currently unknown whether interference participates in spacing functional human initiation events. By using a novel hybridization strategy, genomic Morse code, on single combed DNA molecules from primary keratinocytes, we report the initiation sites present on 1.5 Mb of human chromosome 14q11.2. We confirm that initiation zones are widespread in human cells, map to intergenic regions, and contain sequence motifs found at other mammalian initiation zones. Origins used per cell cycle are less abundant than the potential sites of initiation, and their limited use increases the spacing between initiation events. Between-zone interference decreases in proportion to the distance from the active origin, whereas within-zone interference is 100% efficient. These results identify a hierarchical organization of origin activity in human cells. Functional origins govern the probability that nearby origins will fire in the context of multiple potential start sites of DNA replication, and this is mediated by origin interference.

| S-EPMC1679695 | biostudies-literature

Gram-Positive Bacteria-Like DNA Binding Machineries Involved in Replication Initiation and Termination Mechanisms of Mimivirus.

Project description:The detailed mechanisms of replication initiation, termination and segregation events were not yet known in Acanthamoeba polyphaga mimivirus (APMV). Here, we show detailed bioinformatics-based analyses of chromosomal replication in APMV from initiation to termination mediated by proteins bound to specific DNA sequences. Using GC/AT skew and coding sequence skew analysis, we estimated that the replication origin is located at 382 kb in the APMV genome. We performed homology-modeling analysis of the gamma domain of APMV-FtsK (DNA translocase coordinating chromosome segregation) related to FtsK-orienting polar sequences (KOPS) binding, suggesting that there was an insertion in the gamma domain which maintains the structure of the DNA binding motif. Furthermore, UvrD/Rep-like helicase in APMV was homologous to Bacillus subtilis AddA, while the chi-like quartet sequence 5'-CCGC-3' was frequently found in the estimated ori region, suggesting that chromosomal replication of APMV is initiated via chi-like sequence recognition by UvrD/Rep-like helicase. Therefore, the replication initiation, termination and segregation of APMV are presumably mediated by DNA repair machineries derived from gram-positive bacteria. Moreover, the other frequently observed quartet sequence 5'-CGGC-3' in the ori region was homologous to the mitochondrial signal sequence of replication initiation, while the comparison of quartet sequence composition in APMV/Rickettsia-genome showed significantly similar values, suggesting that APMV also conserves the mitochondrial replication system acquired from an ancestral genome of mitochondria during eukaryogenesis.

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