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DNA replication and transcription occur in all living cells across all domains of life. Both essential processes occur simultaneously on the same template, leading to conflicts between the macromolecular machines that perform these functions. Numerous studies over the past few decades demonstrate that this is an inevitable problem in both prokaryotic and eukaryotic cells. We have learned that conflicts lead to replication fork reversal, breaks in the DNA, R-loop formation, topological stress, and mutagenesis and can ultimately impact evolution. Recent studies have also provided insight into the various mechanisms that mitigate, resolve, and allow tolerance of conflicts and how conflicts result in pathological consequences across divergent species. In this review, we summarize our current knowledge regarding the outcomes of the encounters between replication and transcription machineries and explore how these clashes are dealt with across species. [ABSTRACT FROM AUTHOR]

Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη) were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

Tandem repeats are inherently unstable and exhibit extensive copy number polymorphisms. Despite mounting evidence for their adaptive potential, the mechanisms associated with regulation of the stability and copy number of tandem repeats remain largely unclear. To study copy number variation at tandem repeats, we used two well-studied repetitive arrays in the budding yeast genome, the ribosomal DNA (rDNA) locus, and the copper-inducible CUP1 gene array. We developed powerful, highly sensitive, and quantitative assays to measure repeat instability and copy number and used them in multiple high-throughput genetic screens to define pathways involved in regulating copy number variation. These screens revealed that rDNA stability and copy number are regulated by DNA replication, transcription, and histone acetylation. Through parallel studies of both arrays, we demonstrate that instability can be induced by DNA replication stress and transcription. Importantly, while changes in stability in response to stress are observed within a few cell divisions, a change in steady state repeat copy number requires selection over time. Further, H3K56 acetylation is required for regulating transcription and transcription-induced instability at the CUP1 array, and restricts transcription-induced amplification. Our work suggests that the modulation of replication and transcription is a direct, reversible strategy to alter stability at tandem repeats in response to environmental stimuli, which provides cells rapid adaptability through copy number variation. Additionally, histone acetylation may function to promote the normal adaptive program in response to transcriptional stress. Given the omnipresence of DNA replication, transcription, and chromatin marks like histone acetylation, the fundamental mechanisms we have uncovered significantly advance our understanding of the plasticity of tandem repeats more generally. [ABSTRACT FROM AUTHOR]

Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication–transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution. [ABSTRACT FROM AUTHOR]

Transfer RNA (tRNA) genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae. tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways. [ABSTRACT FROM AUTHOR]

The forkhead box (Fox) transcription factors (TFs) are widespread from yeast to humans. Their mutations and dysregulation have been linked to a broad spectrum of malignant neoplasias. They are known as critical players in DNA repair, metabolism, cell cycle control, differentiation, and aging. Recent studies, especially those from the simple model eukaryotes, revealed unexpected contributions of Fox TFs in chromosome replication and organization. More importantly, besides functioning as a canonical TF in cell signaling cascades and gene expression, Fox TFs can directly participate in DNA replication and determine the global replication timing program in a transcription-independent mechanism. Yeast Fox TFs preferentially recruit the limiting replication factors to a subset of early origins on chromosome arms. Attributed to their dimerization capability and distinct DNA binding modes, Fkh1 and Fkh2 also promote the origin clustering and assemblage of replication elements (replication factories). They can mediate long-range intrachromosomal and interchromosomal interactions and thus regulate the four-dimensional chromosome organization. The novel aspects of Fox TFs reviewed here expand their roles in maintaining genome integrity and coordinating the multiple essential chromosome events. These will inevitably be translated to our knowledge and new treatment strategies of Fox TF-associated human diseases including cancer. [ABSTRACT FROM AUTHOR]

Naturally stalled replication forks are considered to cause structurally abnormal chromosomes in tumor cells. However, underlying mechanisms remain speculative, as capturing naturally stalled forks has been a challenge. Here, we captured naturally stalled forks in tumor cells and delineated molecular processes underlying the structural evolution of circular mini-chromosomes (double-minute chromosomes; DMs). Replication forks stalled on the DM by the co-directional collision with the transcription machinery for long non-coding RNA. RPA, BRCA2, and DNA polymerase eta (Polη) were recruited to the stalled forks. The recruitment of Polη was critical for replication to continue, as Polη knockdown resulted in DM loss. Rescued stalled forks were error-prone and switched replication templates repeatedly to create complex fusions of multiple short genomic segments. In mice, such complex fusions circularized the genomic region surrounding MYC to create a DM during tumorigenesis. Our results define a molecular path that guides stalled replication forks to complex chromosomal rearrangements.

We use genetic assays to suggest that transcription-coupled repair or new origin formation in Escherichia coli involves removal of RNAP to create an RNA primer for DNA synthesis. Transcription factor DksA was shown to play a role in numerous reactions involving RNA polymerase. Some, but not all, of the activities of DksA at promoters or during transcription elongation require (p)ppGpp. In addition to its role during transcription, DksA is also involved in maintaining genome integrity. Cells lacking DksA are sensitive to multiple DNA damaging agents including UV light, ionizing radiation, mitomycin C, and nalidixic acid. Here, we focus on two recent studies addressing the importance of DksA in the repair of double-strand breaks (DSBs), one by Sivaramakrishnan et al. (Nature 550:214–218, 2017) and one originating in our laboratory, Myka et al. (Mol Microbiol 111:1382–1397. 10.1111/mmi.14227, 2019). It appears that depending on the type and possibly location of DNA damage, DksA can play either a passive or an active role in DSB repair. The passive role relies on exclusion of anti-backtracking factors from the RNAP secondary channel. The exact mechanism of active DksA-mediated DNA repair is unknown. However, DksA was proposed to destabilize transcription complexes, thus clearing the way for recombination and DNA repair. Based on the requirement for DksA, both in repair of DSBs and the R-loop-dependent formation of new origins of DNA replication, we propose that DksA may allow for removal of RNAP without unwinding of the RNA:DNA hybrid, which can then be extended by a DNA polymerase. This mechanism obviates the need for RNAP backtracking to repair damaged DNA. [ABSTRACT FROM AUTHOR]

Ribosomes are large, multi-subunit ribonucleoprotein complexes, essential for protein synthesis. To meet the high cellular demand for ribosomes, all eukaryotes have numerous copies of ribosomal DNA (rDNA) genes that encode ribosomal RNA (rRNA), usually far in excess of the requirement for ribosome biogenesis. In all eukaryotes studied, rDNA genes are arranged in one or more clusters of tandem repeats localized to nucleoli. The tandem arrangement of repeats, combined with the high rates of transcription at the rDNA loci, and the difficulty of replicating repetitive sequences make the rDNA inherently unstable and particularly susceptible to large variations in repeat copy number. Despite mounting evidence suggesting extra-ribosomal functions of the rDNA, its repetitive nature has excluded it from traditional sequencing-based studies. However, more recently, several studies have revealed the unique potential of the rDNA to act as a "canary in the coalmine," being particularly sensitive to genomic stresses and acting as a source of adaptive response. Here, we review evidence uncovering mechanisms of regulation of instability and copy number variation at the rDNA and their role in adaptation to the environment, which could serve to understand the basic principles governing the behavior of other tandem repeats and their role in shaping the genome. [ABSTRACT FROM AUTHOR]

Finally, chromosome segregation constitutes an essential stage of cell cycle progression, and ParA and ParB are known as main players in cellular positioning of the replication origin prior to cell division in most bacterial species. Keywords: bacterial chromosomes; nucleoid architecture; DNA replication; chromosome segregation; DNA repair; stress conditions; environmental conditions; replication-transcription conflicts EN bacterial chromosomes nucleoid architecture DNA replication chromosome segregation DNA repair stress conditions environmental conditions replication-transcription conflicts N.PAG N.PAG 3 03/16/21 20210311 NES 210311 The bacterial cell cycle comprises chromosome replication and segregation of newly replicated chromosomes into daughter cells prior to cell division. Bacterial chromosomes, nucleoid architecture, DNA replication, chromosome segregation, DNA repair, environmental conditions, replication-transcription conflicts, stress conditions In this context, Sinha et al. review the current understanding of molecular mechanisms underlying the negative impact of the stringent stress response regulator, (p)ppGpp, on chromosome replication initiation in I Escherichia coli i , and on chromosome replication elongation in I Bacillus subtilis i , respectively. [Extracted from the article]

Highlights • In ΔrecB cells DNA degradation is triggered at forks stalled at rrn operons. • No such degradation is observed in cells lacking Rep helicase. • In ΔrecB cells DNA degradation is triggered in the termination area. • SbcCD is the nuclease mostly responsible for degradation in both scenarios. • In ΔrecB cells degradation can occur at both ends of a linear chromosome. Abstract Bacterial chromosome duplication is initiated at a single origin (oriC). Two forks are assembled and proceed in opposite directions with high speed and processivity until they fuse and terminate in a specialised area opposite to oriC. Proceeding forks are often blocked by tightly-bound protein-DNA complexes, topological strain or various DNA lesions. In Escherichia coli the RecBCD protein complex is a key player in the processing of double-stranded DNA (dsDNA) ends. It has important roles in the repair of dsDNA breaks and the restart of forks stalled at sites of replication-transcription conflicts. In addition, ΔrecB cells show substantial amounts of DNA degradation in the termination area. In this study we show that head-on encounters of replication and transcription at a highly-transcribed rrn operon expose fork structures to degradation by nucleases such as SbcCD. SbcCD is also mostly responsible for the degradation in the termination area of ΔrecB cells. However, additional processes exacerbate degradation specifically in this location. Replication profiles from ΔrecB cells in which the chromosome is linearized at two different locations highlight that the location of replication termination can have some impact on the degradation observed. Our data improve our understanding of the role of RecBCD at sites of replication-transcription conflicts as well as the final stages of chromosome duplication. However, they also highlight that current models are insufficient and cannot explain all the molecular details in cells lacking RecBCD. [ABSTRACT FROM AUTHOR]

Within the last decade, it has become clear that DNA replication and transcription are routinely in conflict with each other in growing cells. Much of the seminal work on this topic has been carried out in bacteria, specifically, Escherichia coli and Bacillus subtilis; therefore, studies of conflicts in these species deserve special attention. Collectively, the recent findings on conflicts have fundamentally changed the way we think about DNA replication in vivo. Furthermore, new insights on this topic have revealed that the conflicts between replication and transcription significantly influence many key parameters of cellular function, including genome organization, mutagenesis, and evolution of stress response and virulence genes. In this review, we discuss the consequences of replication-transcription conflicts on the life of bacteria and describe some key strategies cells use to resolve them. We put special emphasis on two critical aspects of these encounters: (a) the consequences of conflicts on replisome stability and dynamics, and (b) the resulting increase in spontaneous mutagenesis. [ABSTRACT FROM AUTHOR]

Head-on replication–transcription conflict is especially bitter in bacterial chromosomes, explaining why actively transcribed genes are always co-oriented with replication. The mechanism of this conflict remains unclear, besides the anticipated accumulation of positive supercoils between head-on-conflicting polymerases. Unexpectedly, experiments in bacterial and human cells reveal that head-on replication–transcription conflict induces R-loops, indicating hypernegative supercoiling [(−)sc] in the region – precisely the opposite of that assumed. Further, as a result of these R-loops, both replication and transcription in the affected region permanently stall, so the failure of R-loop removal in RNase H-deficient bacteria becomes lethal. How hyper(−)sc emerges in the middle of a positively supercoiled chromosomal domain is a mystery that requires rethinking of topoisomerase action around polymerases. [ABSTRACT FROM AUTHOR]

The canonical model of DNA replication describes a highly-processive and largely continuous process by which the genome is duplicated. This continuous model is based upon in vitro reconstitution and in vivo ensemble experiments. Here, we characterize the replisome-complex stoichiometry and dynamics with single-molecule resolution in bacterial cells. Strikingly, the stoichiometries of the replicative helicase, DNA polymerase, and clamp loader complexes are consistent with the presence of only one active replisome in a significant fraction of cells (>40%). Furthermore, many of the observed complexes have short lifetimes (

Evolution could potentially be accelerated if an organism could selectively increase the mutation rate of specific genes that are actively under positive selection. Recently, a mechanism that cells can use to target rapid evolution to specific genes was discovered. This mechanism is driven by gene orientation-dependent encounters between DNA replication and transcription machineries. These encounters increase mutagenesis in lagging-strand genes, where replication–transcription conflicts are severe. Due to the orientation and transcription-dependent nature of this process, conflict-driven mutagenesis can be used by cells to spatially (gene-specifically) and temporally (only upon transcription induction) regulate the rate of gene evolution. Here, I summarize recent findings on this topic, and discuss the implications of increasing mutagenesis rates and accelerating evolution through active mechanisms. [ABSTRACT FROM AUTHOR]

We previously discovered that lagging strand genes evolve faster in Bacillus subtilis (and potentially other bacteria). Lagging strand genes are transcribed in the head-on orientation with respect to DNA replication, leading to collisions between the two machineries that stall replication and can destabilize genomes. Our previous work indicated that the increased mutagenesis of head-on genes depends on transcription-coupled repair and the activity of an error prone polymerase which is likely activated in response to these collisions. Recently, it was proposed that sequence context is a major contributor to the increased mutagenesis and evolution of head-on genes. These models are based on laboratory-based evolution experiments performed in B. subtilis. However, critical evolutionary analyses of naturally occurring single nucleotide polymorphisms (SNPs) in wild strains were not performed. Using the genomic sequences from nine closely related wild B. subtilis strains, we analyzed over 200,000 naturally occurring SNPs as a proxy for natural mutation patterns for all genes and in particular, head-on genes. Our analysis suggests that (frame-independent) triplet sequence context can impact mutation rates: certain triplet sequences (TAG, CCC, CTA, and ACC) accumulate SNPs at a higher rate and are depleted from the genome. However, the triplet sequences previously identified as mutagenic in laboratory experiments (CCG, GCG, and CAC) do not have an elevated rate of SNP accumulation and are not depleted from the genome. Importantly, dN/dS analyses indicate that the accelerated evolution of head-on genes is not dependent on any particular triplet sequence. Thus, in agreement with our previous results, mutagenic transcription-coupled repair, rather than sequence context, is sufficient to explain the accelerated evolution of head-on genes. [ABSTRACT FROM AUTHOR]

The duplication of genetic information is central to life. The replication of genetic information is strictly controlled to ensure that each piece of genomic DNA is copied only once during a cell cycle. Factors that slow or stop replication forks cause replication stress. Replication stress is a major source of genome instability in cancer cells. Multiple control mechanisms facilitate the unimpeded fork progression, prevent fork collapse and coordinate fork repair. Chromatin alterations, caused by histone post-translational modifications and chromatin remodeling, have critical roles in normal replication and in avoiding replication stress and its consequences. This text reviews the chromatin regulators that ensure DNA replication and the proper response to replication stress. We also briefly touch on exploiting replication stress in therapeutic strategies. As chromatin regulators are frequently mutated in cancer, manipulating their activity could provide many possibilities for personalized treatment., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance. [Display omitted] • Reducing histone H1 content unveils thousands of chromatin-bound regulatory ncRNAs • Upon H1 depletion, ncRNAs are actively transcribed and have reduced levels of m6A • Low m6A levels at ncRNAs cause conflicts with incoming DNA replication forks • Impairing m6A pathway rescues replication-transcription conflicts at H1-depleted cells Fernández-Justel et al. report an unexpected role for histone H1 in the regulation of non-coding RNA production and turnover with important implications for genome maintenance. They uncover a link between chromatin conformation imparted by histone H1 and the m6A gene-regulatory axis. [ABSTRACT FROM AUTHOR]

Encounters between DNA replication and transcription can cause genomic disruption, particularly when the two meet head-on. Whether these conflicts produce point mutations is debated. This paper presents detailed analyses of a large collection of mutations generated during mutation accumulation experiments with mismatch repair (MMR)-defective Escherichia coli. With MMR absent, mutations are primarily due to DNA replication errors. Overall, there were no differences in the frequencies of base pair substitutions or small indels (i.e., insertion and deletions of ≤4 bp) in the coding sequences or promoters of genes oriented codirectionally versus head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for indels in genes oriented head-on to replication, but this difference was almost entirely due to the asymmetrical genomic locations of tRNA genes containing mononucleotide runs, which are hot spots for indels. No additional orientation bias in mutation frequencies occurred when MMR - strains were also defective for transcription-coupled repair (TCR). However, in contrast to other reports, loss of TCR slightly increased the overall mutation rate, meaning that TCR is antimutagenic. There was no orientation bias in mutation frequencies among the stress response genes that are regulated by RpoS or induced by DNA damage. Thus, biases in the locations of mutational targets can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on versus codirectional to replication. In addition, the data revealed a strong correlation of the frequency of base pair substitutions with gene length but no correlation with gene expression levels. IMPORTANCE Because DNA replication and transcription occur on the same DNA template, encounters between the two machines occur frequently. When these encounters are head-to-head, genomic disruption can occur. However, whether replication-transcription conflicts contribute to spontaneous mutations is debated. Analyzing in detail a large collection of mutations generated with mismatch repair-defective Escherichia coli strains, we found that across the genome there are no significant differences in mutation frequencies between genes oriented codirectionally and those oriented head-on to replication. Among a subset of highly expressed genes, there was a 2- to 3-fold bias for small insertions and deletions in head-on-oriented genes, but this difference was almost entirely due to the asymmetrical locations of tRNA genes containing mononucleotide runs, which are hot spots for these mutations. Thus, biases in the positions of mutational target sequences can account for most, if not all, apparent biases in mutation frequencies between genes oriented head-on and codirectionally to replication.

• Both overexpression and depletion of RUVBL1 induce transcription-dependent replication stress, but the underlying mechanisms are different. • RUVBL1 overexpression results in increased c-Myc dependent pause release of RNAPII. • RUVBL1 knock-down leads to accumulation of ubiquitinated RNA polymerases on chromatin, likely due to impaired INO80 remodeler function. Chromatin regulators control transcription and replication, however if and how they might influence the coordination of these processes still is largely unknown. RUVBL1 and the related ATPase RUVBL2 participate in multiple nuclear processes and are implicated in cancer. Here, we report that both the excess and the deficit of the chromatin regulator RUVBL1 impede DNA replication as a consequence of altered transcription. Surprisingly, cells that either overexpressed or were silenced for RUVBL1 had slower replication fork rates and accumulated phosphorylated H2AX, dependent on active transcription. However, the mechanisms of transcription-dependent replication stress were different when RUVBL1 was overexpressed and when depleted. RUVBL1 overexpression led to increased c-Myc-dependent pause release of RNAPII, as evidenced by higher overall transcription, much stronger Ser2 phosphorylation of Rpb1- C-terminal domain, and enhanced colocalization of Rpb1 and c-Myc. RUVBL1 deficiency resulted in increased ubiquitination of Rpb1 and reduced mobility of an RNAP subunit, suggesting accumulation of stalled RNAPIIs on chromatin. Overall, our data show that by modulating the state of RNAPII complexes, RUVBL1 deregulation induces replication-transcription interference and compromises genome integrity during S-phase. [ABSTRACT FROM AUTHOR]

Conflicts between the replication and transcription machineries have profound effects on chromosome duplication, genome organization, and evolution across species. Head-on conflicts (lagging-strand genes) are significantly more detrimental than codirectional conflicts (leading-strand genes). The fundamental reason for this difference is unknown. Here, we report that topological stress significantly contributes to this difference. We find that head-on, but not codirectional, conflict resolution requires the relaxation of positive supercoils by the type II topoisomerases DNA gyrase and Topo IV, at least in the Gram-positive model bacterium Bacillus subtilis. Interestingly, our data suggest that after positive supercoil resolution, gyrase introduces excessive negative supercoils at head-on conflict regions, driving pervasive R-loop formation. Altogether, our results reveal a fundamental mechanistic difference between the two types of encounters, addressing a long-standing question in the field of replication-transcription conflicts., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The bacterium Escherichia coli can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4 to 0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. IMPORTANCE The bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication., (Copyright © 2020 Veetil et al.)

The association between macrocephaly and autism spectrum disorder (ASD) suggests that the mechanisms underlying excessive neural growth could contribute to ASD pathogenesis. Consistently, neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (hiPSCs) of ASD individuals with early developmental brain enlargement are inherently more proliferative than control NPCs. Here, we show that hiPSC-derived NPCs from ASD individuals with macrocephaly display an altered DNA replication program and increased DNA damage. When compared with the control NPCs, high-throughput genome-wide translocation sequencing (HTGTS) demonstrates that ASD-derived NPCs harbored elevated DNA double-strand breaks in replication stress-susceptible genes, some of which are associated with ASD pathogenesis. Our results provide a mechanism linking hyperproliferation of NPCs with the pathogenesis of ASD by disrupting long neural genes involved in cell-cell adhesion and migration., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The forkhead box (Fox) transcription factors (TFs) are widespread from yeast to humans. Their mutations and dysregulation have been linked to a broad spectrum of malignant neoplasias. They are known as critical players in DNA repair, metabolism, cell cycle control, differentiation, and aging. Recent studies, especially those from the simple model eukaryotes, revealed unexpected contributions of Fox TFs in chromosome replication and organization. More importantly, besides functioning as a canonical TF in cell signaling cascades and gene expression, Fox TFs can directly participate in DNA replication and determine the global replication timing program in a transcription-independent mechanism. Yeast Fox TFs preferentially recruit the limiting replication factors to a subset of early origins on chromosome arms. Attributed to their dimerization capability and distinct DNA binding modes, Fkh1 and Fkh2 also promote the origin clustering and assemblage of replication elements (replication factories). They can mediate long-range intrachromosomal and interchromosomal interactions and thus regulate the four-dimensional chromosome organization. The novel aspects of Fox TFs reviewed here expand their roles in maintaining genome integrity and coordinating the multiple essential chromosome events. These will inevitably be translated to our knowledge and new treatment strategies of Fox TF-associated human diseases including cancer.

Conflicts between replication and transcription can have life-threatening consequences. RNA polymerase (RNAP) is the major impediment to replication progression, and its efficient removal from DNA should mitigate the consequences of collisions with replication. Cells have various proteins that can resolve conflicts by removing stalled (or actively translocating) RNAP from DNA. It would therefore seem logical that RNAP-associated factors, such as the bacterial DNA translocase Mfd, would minimize the effects of conflicts. Despite seemingly conclusive statements in most textbooks, the role of Mfd in conflicts remains an enigma. In this review, we will discuss the different physical states of RNAP during transcription, and how each distinct state can influence conflict severity and potentially trigger the involvement of Mfd. We propose models to explain the contradictory conclusions from published studies on the potential role of Mfd in resolving conflicts. [ABSTRACT FROM AUTHOR]

Summary Replication-transcription collisions shape genomes, influence evolution, and promote genetic diseases. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication and promotes genomic instability. Here, we find that head-on collisions promote R-loop formation in Bacillus subtilis . We show that pervasive R-loop formation at head-on collision regions completely blocks replication, elevates mutagenesis, and inhibits gene expression. Accordingly, the activity of the R-loop processing enzyme RNase HIII at collision regions is crucial for stress survival in B. subtilis, as many stress response genes are head-on to replication. Remarkably, without RNase HIII, the ability of the intracellular pathogen Listeria monocytogenes to infect and replicate in hosts is weakened significantly, most likely because many virulence genes are head-on to replication. We conclude that the detrimental effects of head-on collisions stem primarily from excessive R-loop formation and that the resolution of these structures is critical for bacterial stress survival and pathogenesis. [ABSTRACT FROM AUTHOR]

The bacterium Escherichia coli contains a single circular chromosome with a defined architecture. DNA replication initiates at a single origin called oriC. Two replication forks are assembled and proceed in opposite directions until they fuse in a specialised zone opposite the origin. This termination area is flanked by polar replication fork pause sites that allow forks to enter, but not to leave. Thus, the chromosome is divided into two replichores, each replicated by a single replication fork. Recently, we analysed the replication parameters in E. coli cells, in which an ectopic origin termed oriZ was integrated in the right-hand replichore. Two major obstacles to replication were identified: (1) head-on replication⁻transcription conflicts at highly transcribed rrn operons, and (2) the replication fork trap. Here, we describe replication parameters in cells with ectopic origins, termed oriX and oriY , integrated into the left-hand replichore, and a triple origin construct with oriX integrated in the left-hand and oriZ in the right-hand replichore. Our data again highlight both replication⁻transcription conflicts and the replication fork trap as important obstacles to DNA replication, and we describe a number of spontaneous large genomic rearrangements which successfully alleviate some of the problems arising from having an additional origin in an ectopic location. However, our data reveal additional factors that impact efficient chromosome duplication, highlighting the complexity of chromosomal architecture.

The canonical model of DNA replication describes a highly-processive and largely continuous process by which the genome is duplicated. This continuous model is based upon in vitro reconstitution and in vivo ensemble experiments. Here, we characterize the replisome-complex stoichiometry and dynamics with single-molecule resolution in bacterial cells. Strikingly, the stoichiometries of the replicative helicase, DNA polymerase, and clamp loader complexes are consistent with the presence of only one active replisome in a significant fraction of cells (>40%). Furthermore, many of the observed complexes have short lifetimes (<8 min), suggesting that replisome disassembly is quite prevalent, possibly occurring several times per cell cycle. The instability of the replisome complex is conflict-induced: transcription inhibition stabilizes these complexes, restoring the second replisome in many of the cells. Our results suggest that, in contrast to the canonical model, DNA replication is a largely discontinuous process in vivo due to pervasive replication-transcription conflicts., Competing Interests: The authors declare that no competing interests exist.

DNA replication and transcription are vital cellular processes during which the genetic information is copied into complementary DNA and RNA molecules. Highly complex machineries required for DNA and RNA synthesis compete for the same DNA template, therefore being on a collision course. Unscheduled replication-transcription clashes alter the gene transcription program and generate replication stress, reducing fork speed. Molecular pathways and mechanisms that minimize the conflict between replication and transcription have been extensively characterized in prokaryotic cells and recently identified also in eukaryotes. A pathological outcome of replication-transcription collisions is the formation of stable RNA:DNA hybrids in molecular structures called R-loops. Growing evidence suggests that R-loop accumulation promotes both genetic and epigenetic instability, thus severely affecting genome functionality. In the present review, we summarize the current knowledge related to replication and transcription conflicts in eukaryotes, their consequences on genome stability and the pathways involved in their resolution. These findings are relevant to clarify the molecular basis of cancer and neurodegenerative diseases.