Non-cell-autonomous disruption of nuclear architecture as a potential cause of COVID-19-induced anosmia.

SARS-CoV-2 infects less than 1% of cells in the human body, yet it can cause severe damage in a variety of organs. Thus, deciphering the non-cell-autonomous effects of SARS-CoV-2 infection is imperative for understanding the cellular and molecular disruption it elicits. Neurological and cognitive defects are among the least understood symptoms of COVID-19 patients, with olfactory dysfunction being their most common sensory deficit. Here, we show that both in humans and hamsters, SARS-CoV-2 infection causes widespread downregulation of olfactory receptors (ORs) and of their signaling components. This non-cell-autonomous effect is preceded by a dramatic reorganization of the neuronal nuclear architecture, which results in dissipation of genomic compartments harboring OR genes. Our data provide a potential mechanism by which SARS-CoV-2 infection alters the cellular morphology and the transcriptome of cells it cannot infect, offering insight to its systemic effects in olfaction and beyond.

Single-molecule tracking reveals the functional allocation, in vivo interactions, and spatial organization of universal transcription factor NusG.

During transcription elongation, NusG aids RNA polymerase by inhibiting pausing, promoting anti-termination on rRNA operons, coupling transcription with translation on mRNA genes, and facilitating Rho-dependent termination. Despite extensive work, the in vivo functional allocation and spatial distribution of NusG remain unknown. Using single-molecule tracking and super-resolution imaging in live E. coli cells, we found NusG predominantly in a chromosome-associated population (binding to RNA polymerase in elongation complexes) and a slowly diffusing population complexed with the 30S ribosomal subunit; the latter provides a "30S-guided" path for NusG into transcription elongation. Only ∼10% of NusG is fast diffusing, with its mobility suggesting non-specific interactions with DNA for >50% of the time. Antibiotic treatments and deletion mutants revealed that chromosome-associated NusG participates mainly in rrn anti-termination within phase-separated transcriptional condensates and in transcription-translation coupling. This study illuminates the multiple roles of NusG and offers a guide on dissecting multi-functional machines via in vivo imaging.

Mechanisms of insertions at a DNA double-strand break.

Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay called insertion and deletion sequencing (Indel-seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentally induced and spontaneous genome instability. Templated insertions, which derive from sequences genome wide, require contact between donor and acceptor loci, require homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop, followed by DNA synthesis, displacement, and then ligation by non-homologous end joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that is distinct from cut-and-paste events.

Linking RNA polymerase backtracking to genome instability in E. coli.

Frequent codirectional collisions between the replisome and RNA polymerase (RNAP) are inevitable because the rate of replication is much faster than that of transcription. Here we show that, in E. coli, the outcome of such collisions depends on the productive state of transcription elongation complexes (ECs). Codirectional collisions with backtracked (arrested) ECs lead to DNA double-strand breaks (DSBs), whereas head-on collisions do not. A mechanistic model is proposed to explain backtracking-mediated DSBs. We further show that bacteria employ various strategies to avoid replisome collisions with backtracked RNAP, the most general of which is translation that prevents RNAP backtracking. If translation is abrogated, DSBs are suppressed by elongation factors that either prevent backtracking or reactivate backtracked ECs. Finally, termination factors also contribute to genomic stability by removing arrested ECs. Our results establish RNAP backtracking as the intrinsic hazard to chromosomal integrity and implicate active ribosomes and other anti-backtracking mechanisms in genome maintenance.

Nuclear ARP2/3 drives DNA break clustering for homology-directed repair.

DNA double-strand breaks repaired by non-homologous end joining display limited DNA end-processing and chromosomal mobility. By contrast, double-strand breaks undergoing homology-directed repair exhibit extensive processing and enhanced motion. The molecular basis of this movement is unknown. Here, using Xenopus laevis cell-free extracts and mammalian cells, we establish that nuclear actin, WASP, and the actin-nucleating ARP2/3 complex are recruited to damaged chromatin undergoing homology-directed repair. We demonstrate that nuclear actin polymerization is required for the migration of a subset of double-strand breaks into discrete sub-nuclear clusters. Actin-driven movements specifically affect double-strand breaks repaired by homology-directed repair in G2 cell cycle phase; inhibition of actin nucleation impairs DNA end-processing and homology-directed repair. By contrast, ARP2/3 is not enriched at double-strand breaks repaired by non-homologous end joining and does not regulate non-homologous end joining. Our findings establish that nuclear actin-based mobility shapes chromatin organization by generating repair domains that are essential for homology-directed repair in eukaryotic cells.

Activation of DSB processing requires phosphorylation of CtIP by ATR.

DNA double-strand breaks (DSBs) activate a DNA damage response (DDR) that coordinates checkpoint pathways with DNA repair. ATM and ATR kinases are activated sequentially. Homology-directed repair (HDR) is initiated by resection of DSBs to generate 3' single-stranded DNA overhangs. How resection and HDR are activated during DDR is not known, nor are the roles of ATM and ATR in HDR. Here, we show that CtIP undergoes ATR-dependent hyperphosphorylation in response to DSBs. ATR phosphorylates an invariant threonine, T818 of Xenopus CtIP (T859 in human). Nonphosphorylatable CtIP (T818A) does not bind to chromatin or initiate resection. Our data support a model in which ATM activity is required for an early step in resection, leading to ATR activation, CtIP-T818 phosphorylation, and accumulation of CtIP on chromatin. Chromatin binding by modified CtIP precedes extensive resection and full checkpoint activation.

Inorganic phosphate, arsenate, and vanadate enhance exonuclease transcript cleavage by RNA polymerase by 2000-fold.

Inorganic Pi is involved in all major biochemical pathways. Here we describe a previously unreported activity of Pi We show that Pi and its structural mimics, vanadate and arsenate, enhance nascent transcript cleavage by RNA polymerase (RNAP). They engage an Mg2+ ion in catalysis and activate an attacking water molecule. Pi, vanadate, and arsenate stimulate the intrinsic exonuclease activity of the enzyme nearly 2,000-fold at saturating concentrations of the reactant anions and Mg2+ This enhancement is comparable to that of specialized transcript cleavage protein factors Gre and TFIIS (3,000- to 4,000-fold). Unlike these protein factors, Pi and its analogs do not stimulate endonuclease transcript cleavage. Conversely, the protein factors only marginally enhance exonucleolytic cleavage. Pi thus complements cellular protein factors in assisting hydrolytic RNA cleavage by extending the repertoire of RNAP transcript degradation modes.

Ribonucleoside-5'-diphosphates (NDPs) support RNA polymerase transcription, suggesting NDPs may have been substrates for primordial nucleic acid biosynthesis.

A better understanding of the structural basis for the preferences of RNA and DNA polymerases for nucleoside-5'-triphosphates (NTPs) could help define the catalytic mechanisms for nucleotidyl transfer during RNA and DNA synthesis and the origin of primordial nucleic acid biosynthesis. We show here that ribonucleoside-5'-diphosphates (NDPs) can be utilized as substrates by RNA polymerase (RNAP). We found that NDP incorporation is template-specific and that noncognate NDPs are not incorporated. Compared with the natural RNAP substrates, NTPs, the Km of RNAP for NDPs was increased ∼4-fold, whereas the Vmax was decreased ∼200-fold. These properties could be accounted for by molecular modeling of NTP/RNAP co-crystal structures. This finding suggested that the terminal phosphate residue in NTP (not present in NDP) is important for positioning the nucleotide for nucleolytic attack in the nucleotidyl transfer reaction. Strikingly, a mutational substitution of the active-center ßR1106 side chain involved in NTP positioning also strongly inhibited NDP-directed synthesis, even though this residue does not contact NDP. Substitutions in the structurally analogous side chain in RB69 DNA polymerase (Arg-482) and HIV reverse transcriptase (Lys-65) were previously observed to inhibit dNDP incorporation. The unexpected involvement of these residues suggests that they affect a step in catalysis common for nucleic acid polymerases. The substrate activity of NDPs with RNAP along with those reported for DNA polymerases reinforces the hypothesis that NDPs may have been used for nucleic acid biosynthesis by primordial enzymes, whose evolution then led to the use of the more complex triphosphate derivatives.

DksA-RNA polymerase interactions support new origin formation and DNA repair in Escherichia coli.

The formation of new replication origins (cSDR) and repair of DNA double-strand breaks (DSBs) in E. coli share a commonality. We find that the two processes require the RNAP-associated factor, DksA. However, whereas cSDR also relies on (p)ppGpp, the alarmone molecule is dispensable for the repair of topoisomerase type II (Top II) DNA adducts and associated DSBs. The requirement for DksA in repair of nalidixic acid (Nal)-induced DSBs or for the formation of new origins is not suppressed by a greA deletion mutation, indicating an active role of DksA rather than competition with GreA for insertion into the RNAP secondary channel. Like dksA mutations, transcription termination factor Rho mutations also confer sensitivity to Nal. The rho and dksA mutations are not epistatic, suggesting they involve different repair pathways. The roles of DksA in DSB repair and cSDR differ; certain DksA and RNAP mutants are able to support the first process, but not the latter. We suggest that new origin formation and DNA repair of protein adducts with DSBs may both involve the removal of RNAP without destruction of the RNA:DNA hybrid.

Replication-independent repair of DNA interstrand crosslinks.

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that covalently link opposite strands of the DNA helix and block DNA unwinding. ICLs are repaired during and outside S phase, and replication-independent ICL repair (RIR) is critical to maintain genomic integrity and to allow transcription in nondividing or slowly dividing cells. Here, we show that the Y family DNA polymerase kappa (Pol κ) is essential for RIR of a site-specific ICL lesion in Xenopus egg extracts, and that both its catalytic activity and UBZ domains are required for this function. We also demonstrate a requirement for PCNA and its modification on lysine 164. Finally, we show that Pol κ participates in ICL repair in mammalian cells, particularly in G0. Our results identify key components of the RIR pathway and begin to unravel its mechanism.

Correction: DNA Damage, Homology-Directed Repair, and DNA Methylation.

[This corrects the article DOI: 10.1371/journal.pgen.0030110.].

Escherichia coli transcription factor NusG binds to 70S ribosomes.

Transcription and translation are coupled processes in bacteria. A role of transcription elongation cofactor NusG in coupling has been suggested by in vitro structural studies. NMR revealed association of the NusG carboxy-terminal domain with S10 (NusE), implying a direct role for NusG as a bridge linking RNAP and the lead ribosome. Here we present the first in vitro and in vivo evidence of full-length NusG association with mature 70S ribosomes. Binding did not require accessory factors in vitro. Mutating the NusG:S10 binding interface at NusG F165 or NusE M88 and D97 residues weakened NusG:S10 association in vivo and completely abolished it in vitro, supporting the specificity of this interaction. Mutations in the binding interface increased sensitivity to chloramphenicol. This phenotype was suppressed by rpoB*35, an RNAP mutation that reduces replisome-RNAP clashes. We propose that weakened NusG:S10 interaction leads to uncoupling when translation is inhibited, with resulting RNAP backtracking, replication blocks and formation of lethal DNA double-strand breaks.

DksA and DNA double-strand break repair.

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. https://doi.org/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.

Probing the structure of Nun transcription arrest factor bound to RNA polymerase.

The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5' and the 3' ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3' end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5' end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors.

The differences between ICL repair during and outside of S phase.

DNA interstrand crosslinks (ICLs) are complex lesions that block essential DNA transactions including DNA replication, recombination, and RNA transcription. Naturally occurring ICLs are rare, yet these lesions are the major cause of toxicity following treatment with several classes of crosslinking cancer chemotherapeutic drugs. ICLs are repaired during and outside of S phase by pathways with overlapping as well as distinct features. Here, we discuss some recent insights into the mechanisms of replication-dependent and replication-independent repair of ICLs with special emphasis on the differences between these repair pathways.

Checkpoint signaling from a single DNA interstrand crosslink.

DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited, because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from, and repairing of a single site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivity to crosslinking agents results from both checkpoint and DNA repair defects.

Structural and functional analysis of the E. coli NusB-S10 transcription antitermination complex.

Protein S10 is a component of the 30S ribosomal subunit and participates together with NusB protein in processive transcription antitermination. The molecular mechanisms by which S10 can act as a translation or a transcription factor are not understood. We used complementation assays and recombineering to delineate regions of S10 dispensable for antitermination, and determined the crystal structure of a transcriptionally active NusB-S10 complex. In this complex, S10 adopts the same fold as in the 30S subunit and is blocked from simultaneous association with the ribosome. Mass spectrometric mapping of UV-induced crosslinks revealed that the NusB-S10 complex presents an intermolecular, composite, and contiguous binding surface for RNAs containing BoxA antitermination signals. Furthermore, S10 overproduction complemented a nusB null phenotype. These data demonstrate that S10 and NusB together form a BoxA-binding module, that NusB facilitates entry of S10 into the transcription machinery, and that S10 represents a central hub in processive antitermination.

Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops.

Histone methylation changes and formation of chromatin loops involving enhancers, promoters and 3' end regions of genes have been variously associated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A receptor, at the RARE-promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA exposure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethylase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elicits an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nucleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5' transcription start site and the 3' end of the genes. The RARE bound-receptor governs the 5' and 3' end selection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription.

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.

Nus transcription elongation factors and RNase III modulate small ribosome subunit biogenesis in Escherichia coli.

Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.