Transcription-Replication Conflicts as a Source of Genome Instability.

Transcription and replication both require large macromolecular complexes to act on a DNA template, yet these machineries cannot simultaneously act on the same DNA sequence. Conflicts between the replication and transcription machineries (transcription-replication conflicts, or TRCs) are widespread in both prokaryotes and eukaryotes and have the capacity to both cause DNA damage and compromise complete, faithful replication of the genome. This review will highlight recent studies investigating the genomic locations of TRCs and the mechanisms by which they may be prevented, mitigated, or resolved. We address work from both model organisms and mammalian systems but predominantly focus on multicellular eukaryotes owing to the additional complexities inherent in the coordination of replication and transcription in the context of cell type-specific gene expression and higher-order chromatin organization.

Hold on Tight: Lagging-Strand DNA Polymerases Synthesize Multiple Okazaki Fragments without Letting Go.

Kapadia et al. (2020) use an innovative single-molecule imaging approach in yeast cells to measure chromatin association of individual replisome subunits, thereby challenging the notion that lagging-strand DNA polymerases frequently dissociate from replisomes during DNA replication in vivo.

Limiting DNA polymerase delta alters replication dynamics and leads to a dependence on checkpoint activation and recombination-mediated DNA repair.

DNA polymerase delta (Pol δ) plays several essential roles in eukaryotic DNA replication and repair. At the replication fork, Pol δ is responsible for the synthesis and processing of the lagging-strand. At replication origins, Pol δ has been proposed to initiate leading-strand synthesis by extending the first Okazaki fragment. Destabilizing mutations in human Pol δ subunits cause replication stress and syndromic immunodeficiency. Analogously, reduced levels of Pol δ in Saccharomyces cerevisiae lead to pervasive genome instability. Here, we analyze how the depletion of Pol δ impacts replication origin firing and lagging-strand synthesis during replication elongation in vivo in S. cerevisiae. By analyzing nascent lagging-strand products, we observe a genome-wide change in both the establishment and progression of replication. S-phase progression is slowed in Pol δ depletion, with both globally reduced origin firing and slower replication progression. We find that no polymerase other than Pol δ is capable of synthesizing a substantial amount of lagging-strand DNA, even when Pol δ is severely limiting. We also characterize the impact of impaired lagging-strand synthesis on genome integrity and find increased ssDNA and DNA damage when Pol δ is limiting; these defects lead to a strict dependence on checkpoint signaling and resection-mediated repair pathways for cellular viability.

Separable, Ctf4-mediated recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation.

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, and that both processes are disrupted at similar Pol α concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol α, lagging-strand initiation is impaired at Pol α concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α. Using strains in which the Pol α-Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol α recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol α at the replication fork, at least when Pol α is limiting.

Quantitative, genome-wide analysis of eukaryotic replication initiation and termination.

Many fundamental aspects of DNA replication, such as the exact locations where DNA synthesis is initiated and terminated, how frequently origins are used, and how fork progression is influenced by transcription, are poorly understood. Via the deep sequencing of Okazaki fragments, we comprehensively document replication fork directionality throughout the S. cerevisiae genome, which permits the systematic analysis of initiation, origin efficiency, fork progression, and termination. We show that leading-strand initiation preferentially occurs within a nucleosome-free region at replication origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing. The replication profile is predominantly determined by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture.

Processing of eukaryotic Okazaki fragments by redundant nucleases can be uncoupled from ongoing DNA replication in vivo.

Prior to ligation, each Okazaki fragment synthesized on the lagging strand in eukaryotes must be nucleolytically processed. Nuclease cleavage takes place in the context of 5' flap structures generated via strand-displacement synthesis by DNA polymerase delta. At least three DNA nucleases: Rad27 (Fen1), Dna2 and Exo1, have been implicated in processing Okazaki fragment flaps. However, neither the contributions of individual nucleases to lagging-strand synthesis nor the structure of the DNA intermediates formed in their absence have been fully defined in vivo. By conditionally depleting lagging-strand nucleases and directly analyzing Okazaki fragments synthesized in vivo in Saccharomyces cerevisiae, we conduct a systematic evaluation of the impact of Rad27, Dna2 and Exo1 on lagging-strand synthesis. We find that Rad27 processes the majority of lagging-strand flaps, with a significant additional contribution from Exo1 but not from Dna2. When nuclease cleavage is impaired, we observe a reduction in strand-displacement synthesis as opposed to the widespread generation of long Okazaki fragment 5' flaps, as predicted by some models. Further, using cell cycle-restricted constructs, we demonstrate that both the nucleolytic processing and the ligation of Okazaki fragments can be uncoupled from DNA replication and delayed until after synthesis of the majority of the genome is complete.

Intrinsic coupling of lagging-strand synthesis to chromatin assembly.

Fifty per cent of the genome is discontinuously replicated on the lagging strand as Okazaki fragments. Eukaryotic Okazaki fragments remain poorly characterized and, because nucleosomes are rapidly deposited on nascent DNA, Okazaki fragment processing and nucleosome assembly potentially affect one another. Here we show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the nucleosome repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur near nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging-strand polymerase processivity affects both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of Okazaki fragment synthesis. Our studies represent the first high-resolution analysis--to our knowledge--of eukaryotic Okazaki fragments in vivo, and reveal the interconnection between lagging-strand synthesis and chromatin assembly.

Insights into branch nucleophile positioning and activation from an orthogonal pre-mRNA splicing system in yeast.

The duplex formed between the branch site (BS) of a spliceosomal intron and its cognate sequence in U2 snRNA is important for spliceosome assembly and the first catalytic step of splicing. We describe the development of an orthogonal BS-U2 system in S. cerevisiae in which spliceosomes containing a grossly substituted second-copy U2 snRNA mediate the in vivo splicing of a single reporter transcript carrying a cognate substitution. Systematic use of this approach to investigate requirements for branching catalysis reveals considerable flexibility in the sequence of the BS-U2 duplex and its positioning relative to the catalytic center. Branching efficiency depends on the identity of the branch nucleotide, its position within the BS-U2 duplex, and its distance from U2/U6 helix Ia. These results provide insights into substrate selection during spliceosomal branching catalysis; additionally, this system provides a foundation and tool for future mechanistic splicing research.

"Nought may endure but mutability": spliceosome dynamics and the regulation of splicing.

The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing, or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction.

Use of a single parameter track and trigger chart and the perceived barriers and facilitators to escalation of a deteriorating ward patient: a mixed methods study.

AIMS AND OBJECTIVES: To investigate nurses' use of a single parameter track and trigger chart to inform implementation of the National Early Warning Scoring tool. To report the characteristics of patients with triggers, the frequency of different triggers, and the time taken to repeat observations. To explore the barriers and facilitators perceived by nursing staff relating to patient monitoring. BACKGROUND: Sub-optimal care of the deteriorating patient has been described for almost two decades. Organisations have responded by implementing strategies that improve monitoring and facilitate a timely response to patient deterioration. While these systems have been widely adopted the evidence-base to support their use is inconsistent. DESIGN: A mixed method service evaluation was carried out in an acute University hospital. METHODS: Physiological triggers (n = 263) and characteristics of triggering patients (n = 74) were recorded from surgical and medical wards. Descriptive statistics were displayed. Questionnaires were distributed (n = 105) to student nurses, health care assistants and registered nurses. Themes and sub-themes were identified from content analysis. RESULTS: Hypotension was the most frequent abnormality. There was variability in the time to repeat observations following a trigger. A high proportion of triggers were identified in older patients, as was a trend of longer time intervals between trigger and repeat observations. Nurses reported a number of barriers and facilitators to monitoring patients including: 'workload', 'equipment', 'interactions between staff' and 'interactions with patients'. CONCLUSIONS: This study identified a number of barriers and facilitators to monitoring and escalation of abnormal vital signs, highlighting the complexity of the process and the need for a system-wide approach to a deteriorating patient. RELEVANCE TO CLINICAL PRACTICE: The trend of longer delays following a trigger in older patients has not been identified previously and could reflect a knowledge gap of the physiological changes and response to acute illness in older people.

An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae.

Cohesion between sister chromatids, mediated by the chromosomal cohesin complex, is a prerequisite for their alignment on the spindle apparatus and segregation in mitosis. Budding yeast cohesin first associates with chromosomes in G1. Then, during DNA replication in S-phase, the replication fork-associated acetyltransferase Eco1 acetylates the cohesin subunit Smc3 to make cohesin's DNA binding resistant to destabilization by the Wapl protein. Whether stabilization of cohesin molecules that happen to link sister chromatids is sufficient to build sister chromatid cohesion, or whether additional reactions are required to establish these links, is not known. In addition to Eco1, several other factors contribute to cohesion establishment, including Ctf4, Ctf18, Tof1, Csm3, Chl1 and Mrc1, but little is known about their roles. Here, we show that each of these factors facilitates cohesin acetylation. Moreover, the absence of Ctf4 and Chl1, but not of the other factors, causes a synthetic growth defect in cells lacking Eco1. Distinct from acetylation defects, sister chromatid cohesion in ctf4Δ and chl1Δ cells is not improved by removing Wapl. Unlike previously thought, we do not find evidence for a role of Ctf4 and Chl1 in Okazaki fragment processing, or of Okazaki fragment processing in sister chromatid cohesion. Thus, Ctf4 and Chl1 delineate an additional acetylation-independent pathway that might hold important clues as to the mechanism of sister chromatid cohesion establishment.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency.

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function.

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.

USP1-trapping lesions as a source of DNA replication stress and genomic instability.

The deubiquitinase USP1 is a critical regulator of genome integrity through the deubiquitylation of Fanconi Anemia proteins and the DNA replication processivity factor, proliferating cell nuclear antigen (PCNA). Uniquely, following UV irradiation, USP1 self-inactivates through autocleavage, which enables its own degradation and in turn, upregulates PCNA monoubiquitylation. However, the functional role for this autocleavage event during physiological conditions remains elusive. Herein, we discover that cells harboring an autocleavage-defective USP1 mutant, while still able to robustly deubiquitylate PCNA, experience more replication fork-stalling and premature fork termination events. Using super-resolution microscopy and live-cell single-molecule tracking, we show that these defects are related to the inability of this USP1 mutant to be properly recycled from sites of active DNA synthesis, resulting in replication-associated lesions. Furthermore, we find that the removal of USP1 molecules from DNA is facilitated by the DNA-dependent metalloprotease Spartan to counteract the cytotoxicity caused by "USP1-trapping". We propose a utility of USP1 inhibitors in cancer therapy based on their ability to induce USP1-trapping lesions and consequent replication stress and genomic instability in cancer cells, similar to how non-covalent DNA-protein crosslinks cause cytotoxicity by imposing steric hindrances upon proteins involved in DNA transactions.

Identification and characterization of a short 2'-3' bond-forming ribozyme.

A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 2'-3' phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.

A critical assessment of the utility of protein-free splicing systems.

U2 and U6 snRNAs form part of the catalytic spliceosome and represent strong candidates for components of its active site. Over the past decade it has become clear that these snRNAs are capable of catalyzing several different chemical reactions, leading to the widespread conclusion that the spliceosome is a ribozyme. Here, we discuss the advances in both protein-free and fully spliceosomal systems that would be required to conclude that the reactions observed to be catalyzed by protein-free snRNAs are related to splicing and question the reliability of snRNA-only systems as tools for mechanistic splicing research.

Post-replicative nick translation occurs on the lagging strand during prolonged depletion of DNA ligase I in Saccharomyces cerevisiae.

During lagging-strand synthesis, strand-displacement synthesis by DNA polymerase delta (Pol ∂), coupled to nucleolytic cleavage of DNA flap structures, produces a nick-translation reaction that replaces the DNA at the 5' end of the preceding Okazaki fragment. Previous work following depletion of DNA ligase I in Saccharomyces cerevisae suggests that DNA-bound proteins, principally nucleosomes and the transcription factors Abf1/Rap1/Reb1, pose a barrier to Pol ∂ synthesis and thereby limit the extent of nick translation in vivo. However, the extended ligase depletion required for these experiments could lead to ongoing, non-physiological nick translation. Here, we investigate nick translation by analyzing Okazaki fragments purified after transient nuclear depletion of DNA ligase I in synchronized or asynchronous Saccharomyces cerevisiae cultures. We observe that, even with a short ligase depletion, Okazaki fragment termini are enriched around nucleosomes and Abf1/Reb1/Rap1-binding sites. However, protracted ligase depletion leads to a global change in the location of these termini, moving them toward nucleosome dyads from a more upstream location and further enriching termini at Abf1/Reb1/Rap1-binding sites. In addition, we observe an under-representation of DNA derived from DNA polymerase alpha-the polymerase that initiates Okazaki fragment synthesis-around the sites of Okazaki termini obtained from very brief ligase depletion. Our data suggest that, while nucleosomes and transcription factors do limit strand-displacement synthesis by Pol ∂ in vivo, post-replicative nick translation can occur at unligated Okazaki fragment termini such that previous analyses represent an overestimate of the extent of nick translation occurring during normal lagging-strand synthesis.

Monitoring genome-wide replication fork directionality by Okazaki fragment sequencing in mammalian cells.

The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.

Mechanistic insights from reversible splicing catalysis.

Recent work demonstrating the ability of spliceosomes purified after the second catalytic step of splicing to efficiently reverse both steps of the reaction provides answers to several unresolved questions regarding the splicing reaction, and raises many more.

Determinants of Replication-Fork Pausing at tRNA Genes in Saccharomyces cerevisiae.

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.