Biology subjects

Lovett, S. T.

Publications and source records attributed to Lovett, S. T..

7 recordsLinked to original sources

bioRxiv2026

Escherichia coli transcription factors RapA and SspA play opposing roles in tolerance to replication/transcription conflicts after DNA damage

The ability to clear transcription complexes from DNA is especially important after DNA damage that produces replication stress. Using the bacterium E. coli, we show here that mutations in RNA polymerase that reduce termination, inhibitors of Rho-dependent termination, and inversion of a highly transcribed ribosomal RNA operon both enhance sensitivity to the quinolone ciprofloxacin (CPX); and identify two transcription factors, SspA and RapA, that impact these effects in opposite ways.We demonstrate that the rapA promoter is induced by CPX, independent of the LexA/RecA SOS response but is dependent on DnaA. Previous work has shown that RapA is expressed highest in rapidly growing cells whereas SspA levels respond to starvation.The factors have opposing effects on tolerance to chronic exposure to CPX, with RapA promoting cell growth and SspA inhibiting it. Functional SspA is also required for the CPX toxicity of the rRNA operon inversion; in sspA{Delta} mutants it has no negative consequence. In otherwise wild-type cells, loss of RapA has little effect except in strains lacking RNase HI, the enzyme that removes RNA/DNA hybrids from DNA. However, in cells lacking SspA, RapA strongly promotes survival, suggesting that SspA must block positive effects of RapA on tolerance. The RapA requirement for CPX tolerance is not relieved by RNase HI overexpression and therefore RapA must be not be merely preventing R-loop formation. RapA also in some way promotes the use of RNA loops to initiate DNA replication in the absence of DnaA. We propose that SspA stabilizes stalled or post-termination RNAP/DNA complexes and that the presence of SspA prevents RapA release of these complexes.

molecular biology↗

bioRxiv2025

A Novel {psi}-χ Fusion Protein for Unravelling the Contributions of χ to DNA Replication and Repair

Faithful DNA replication in Escherichia coli requires the DNA polymerase III holoenzyme (DNA pol III HE) and its clamp loader complex, which couples processive DNA synthesis with {beta}-clamp loading. The clamp loader accessory subunits {chi} and {psi} link single-stranded DNA-binding protein (SSB) to the replisome, stabilizing replication on SSB-coated templates through interactions with the SSB C-terminal tail. Chi has also been implicated in tolerance to the chain-terminating nucleoside analog azidothymidine (AZT), though whether this function depends on {chi} within DNA pol III HE or on its independent interaction with the YoaA helicase remains unclear. To address this, we engineered {psi}-{chi} fusion proteins with flexible glycine-serine linkers to tether the two subunits while preserving folding and activity. Both fusions were biochemically competent, supporting ATP hydrolysis and clamp loading on SSB-coated DNA. In vivo, however, neither the {psi}-GS12-{chi} fusion nor expression of a {psi}{chi} operon restored AZT tolerance in {Delta}holC cells, whereas expression of {chi} alone was sufficient. Fusion expression impaired growth in both wild-type (WT) and {Delta}holC backgrounds, a phenotype alleviated by disrupting {chi}-SSB binding. These findings support a model in which {chi} must dynamically engage SSB and YoaA outside of the clamp loader to promote AZT tolerance, highlighting the importance of regulated {chi}-SSB interactions in genome maintenance.

biochemistry↗

bioRxiv2023

Spontaneous and double-strand break repair-associated quasipalindrome and frameshift mutagenesis in budding yeast: role of mismatch repair

Although gene conversion (GC) in Saccharomyces cerevisiae is the most error-free way to repair double-strand breaks (DSBs), the mutation rate during homologous recombination is 1000 times greater than during replication. Many mutations involve dissociating a partially-copied strand from its repair template and re-aligning with the same or another template, leading to -1 frameshifts in homonucleotide runs, quasipalindrome (QP)-associated mutations and microhomology-mediated interchromosomal template switches. We studied GC induced by HO endonuclease cleavage at MAT, repaired by an HMR::Kl-URA3 donor. We inserted into HMR::Kl-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield mat::Kl-ura3::QP+4 (Ura-) outcomes, but template-switching produces Ura+ colonies, losing the 4-bp insertion. If the QP arm without the insertion is first encountered by repair DNA polymerase and is then (mis)used as a template, the palindrome is perfected. When the QP+4 arm is encountered first, Ura+ derivatives only occur after second-end capture and second-strand synthesis. QP+4 mutations are suppressed by mismatch repair (MMR) proteins Msh2, Msh3, and Mlh1, but not Msh6. Deleting Rdh54 significantly reduces QP mutations only when events creating Ura+ occur in the context of a D-loop but not during second-strand synthesis. A similar bias is found with a proofreading-defective DNA polymerase mutation (pol3-01). DSB-induced mutations differed in several genetic requirements from spontaneous events. We also created a +1 frameshift in the donor, expanding a run of 4 Cs to 5 Cs. Again, Ura3+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

genetics↗

bioRxiv2021

DnaA and SspA Regulation of the iraD gene of E. coli: an alternative DNA damage response independent of LexA/RecA

The transcription factor RpoS ({sigma}S) of Escherichia coli controls a large number of genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by AZT. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp, and regulated by RIDA (regulatory inactivation of DnaA), dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced RIDA. We demonstrate that the RNA-polymerase associated factor, SspA (stringent starvation protein A), induced by the accumulation of ppGpp, also affects IraD expression, with a positive effect on constitutive expression and a negative effect on AZT-induced expression, in a fashion independent of DnaA. SIGNIFICANCEDNA damage can lead to cell death or genomic instability. Cells have evolved transcriptional responses that sense DNA damage and up-regulate tolerance and repair factors; the LexA/RecA-regulated SOS response in E. coli was the first example of such a system. This work describes an alternative DNA damage response, controlled by DnaA and the IraD post-translational regulator of RpoS. The cellular signals for this response, we propose, are empty replication processivity ({beta}) clamps that accumulate at replication blocks. IraD expression is also regulated by stringent starvation protein, SspA, induced by nutrient deprivation. This SOS-independent DNA damage response integrates a signal of incomplete replication with starvation to modulate expression of genes that promote the completion of replication.

genetics↗

bioRxiv2021

Mutational analysis of DinG-family helicase YoaA: its expression and interaction with replication clamp-loader protein HolC in E. coli

The XP-D/DinG family of DNA helicases contribute to genomic stability in all three domains of life. We investigate here the role of one of these proteins,YoaA, of Escherichia coli. In E. coli,YoaA aids tolerance to the nucleoside azidothymidine (AZT), a DNA replication inhibitor and physically interacts with a subunit of the DNA polymerase III holoenzyme, HolC. We map the residues of YoaA required for HolC interaction to its C-terminus by yeast two-hybrid analysis. We propose that this interaction competes with HolCs interaction with HolD and the rest of the replisome;YoaA indeed inhibits growth when overexpressed, dependent on this interaction region. By gene fusions we show YoaA is repressed by LexA and induced in response to DNA damage as part of the SOS response. Induction of YoaA by AZT is biphasic with an immediate response after treatment and a slower response that peaks in the late log phase of growth. This growth-phase dependent induction by AZT is not blocked by lexA3 (Ind-), which normally negates its self-cleavage, implying another means to induce the DNA damage response that responds to the nutritional state of the cell. We propose that YoaA helicase activity increases access to the 3 nascent strand during replication; consistent with this,YoaA appears to aid removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. YoaA and its paralog DinG also may initiate template-switching that leads to deletions between tandem repeats in DNA. IMPORTANCEMaintaining genomic stability is crucial for all living organisms. Replication of DNA frequently encounters barriers that must be removed to complete genome duplication. Balancing DNA synthesis with its repair is critical and not entirely understood at a mechanistic level.The YoaA protein, studied here, is required for certain types of DNA repair and interacts in an alternative manner with proteins that catalyze DNA replication. YoaA is part of the well-studied LexA-regulated response to DNA damage, the SOS response. We describe an unusual feature of its regulation that promotes induction after DNA damage as the culture begins to experience starvation. Replication fork repair integrates both DNA damage and nutritional signals. We also show that YoaA affects genomic stability.

genetics↗

bioRxiv2020

The role of replication clamp-loader protein HolC of Escherichia coli in overcoming replication /transcription conflicts

In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits as well as the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as x) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study, to elucidate the role of HolC in replication, we isolate spontaneous suppressor mutants in a holC{Delta} strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains or sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (), RpoB ({beta}) and RpoC ({beta}) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. Mfd had no effect nor did elongation factors GreA and GreB. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA rpoC and sspA.These results are consistent with the hypothesis that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity. IMPORTANCETranscription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of E. coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps resolve codirectional collisions between replication and transcription complexes, which become toxic in HolCs absence.

genetics↗

bioRxiv2020

DNA damage-signaling, homologous recombination and genetic mutation induced by 5-azacytidine and DNA-protein crosslinks in Escherichia coli

Covalent linkage between DNA and proteins produces highly toxic lesions and can be caused by commonly used chemotherapeutic agents, by internal and external chemicals and by radiation. In this study, using Escherichia coli, we investigate the consequences of 5-azacytidine (5-azaC), which traps covalent complexes between itself and the Dcm cytosine methyltransferase protein. DNA protein crosslink-dependent effects can be ascertained by effects that arise in wild-type but not in dcm{Delta} strains. We find that 5-azaC induces the bacterial DNA damage response and stimulates homologous recombination, a component of which is Dcm-dependent. Template-switching at an imperfect inverted repeat ("quasipalindrome", QP) is strongly enhanced by 5-azaC and this enhancement was entirely Dcm-dependent. The SOS response helps ameliorate the mutagenic effect of 5-azaC but unbalanced expression of the SOS-induced DNA polymerases, especially PolIV, stimulates QP-associated mutagenesis. In the absence of Lon protease, Dcm-dependent QP-mutagenesis is elevated, suggesting it may play a role in 5-azaC tolerance. Deletions at short tandem repeats, which occur likewise by a replication template-switch, are elevated, but only modestly, by 5-azaC. We see evidence for Dcm-dependent and-independent killing by 5-azaC in sensitive mutants, such as recA, recB, and lon; homologous recombination and deletion mutations are also stimulated in part by a Dcm-independent effect of 5-azaC. Whether this occurs by a different protein/DNA crosslink or by an alternative form of DNA damage is unknown. HighlightsO_LI5-azacytidine is broadly mutagenic and recombinogenic C_LIO_LIIn E. coli, 5-azaC promotes genetic instability through Dcm methyltransferase. C_LIO_LIThere are other, unknown lesions induced by 5-azaC besides Dcm/DNA crosslinks C_LIO_LI5-azaC induces the SOS response, protecting cells from killing and genetic instability C_LI

genetics↗