The DNA damage response of Escherichia coli, revisited: Differential gene expression after replication inhibition.

In 1967, in this journal, Evelyn Witkin proposed the existence of a coordinated DNA damage response in Escherichia coli, which later came to be called the "SOS response." We revisited this response using the replication inhibitor azidothymidine (AZT) and RNA-Seq analysis and identified several features. We confirm the induction of classic Save our ship (SOS) loci and identify several genes, including many of the pyrimidine pathway, that have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that the transcription factor "stringent starvation protein" SspA is as important as LexA in the regulation of AZT-induced genes and that the genes activated by SspA change dramatically after AZT exposure. Our experiments identify additional LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD antiadaptor protein is induced independent of LexA and that a second antiadaptor, IraM is likewise strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these antiadaptor proteins is an integral part of replication stress tolerance.

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 1,000 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 MATα, repaired by an HMR::KI-URA3 donor. We inserted into HMR::KI-URA3 an 18-bp inverted repeat where one arm had a 4-bp insertion. Most GCs yield MAT::KI-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 (poI3-01). 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, Ura+ recombinants markedly increased by disabling MMR, suggesting that MMR acts during GC but favors the unbroken, template strand.

Generation and Repair of Postreplication Gaps in Escherichia coli.

When replication forks encounter template lesions, one result is lesion skipping, where the stalled DNA polymerase transiently stalls, disengages, and then reinitiates downstream to leave the lesion behind in a postreplication gap. Despite considerable attention in the 6 decades since postreplication gaps were discovered, the mechanisms by which postreplication gaps are generated and repaired remain highly enigmatic. This review focuses on postreplication gap generation and repair in the bacterium Escherichia coli. New information to address the frequency and mechanism of gap generation and new mechanisms for their resolution are described. There are a few instances where the formation of postreplication gaps appears to be programmed into particular genomic locations, where they are triggered by novel genomic elements.

Characterization of the Escherichia coli XPD/Rad3 iron-sulfur helicase YoaA in complex with the DNA polymerase III clamp loader subunit chi (χ).

Escherichia coli YoaA aids in the resolution of DNA damage that halts DNA synthesis in vivo in conjunction with χ, an accessory subunit of DNA polymerase III. YoaA and χ form a discrete complex separate from the DNA polymerase III holoenzyme, but little is known about how YoaA and χ work together to help the replication fork overcome damage. Although YoaA is predicted to be an iron-sulfur helicase in the XPD/Rad3 helicase family based on sequence analysis, the biochemical activities of YoaA have not been described. Here, we characterize YoaA and show that purified YoaA contains iron. YoaA and χ form a complex that is stable through three chromatographic steps, including gel filtration chromatography. When overexpressed in the absence of χ, YoaA is mostly insoluble. In addition, we show the YoaA-χ complex has DNA-dependent ATPase activity. Our measurement of the YoaA-χ helicase activity illustrates for the first time YoaA-χ translocates on ssDNA in the 5' to 3' direction and requires a 5' single-stranded overhang, or ssDNA gap, for DNA/DNA unwinding. Furthermore, YoaA-χ preferentially unwinds forked duplex DNA that contains both 3' and 5' single-stranded overhangs versus duplex DNA with only a 5' overhang. Finally, we demonstrate YoaA-χ can unwind damaged DNA that contains an abasic site or damage on 3' ends that stall replication extension. These results are the first biochemical evidence demonstrating YoaA is a bona fide iron-sulfur helicase, and we further propose the physiologically relevant form of the helicase is YoaA-χ.

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

The transcription factor RpoS of Escherichia coli controls many 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 azidothymidine. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at -81 bp and are regulated by 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 regulatory inactivation of DnaA. We also demonstrate that the RNA-polymerase associated factor, 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 azidothymidine-induced expression.

Genetic Analysis of DinG Family Helicase YoaA and Its Interaction with Replication Clamp Loader Protein HolC in Escherichia coli.

The XP-D/DinG family of DNA helicases contributes to genomic stability in all three domains of life. Here, we investigate the role of one of these proteins, YoaA, of Escherichia coli. In E. coli, YoaA aids in 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 HolC's 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 that 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 in the removal of potential A-to-T transversion mutations in ndk mutants, which are prone to nucleotide misincorporation. We provide evidence that YoaA and its paralog DinG may also initiate template switching that leads to deletions between tandem repeats in DNA. IMPORTANCE Maintaining 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.

DNA polymerase III protein, HolC, helps resolve 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, the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as χ) 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 we isolate spontaneous suppressor mutants in a holCΔ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, and of 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 (α), RpoB (ß) and RpoC (ß') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. 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. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose 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.

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, the processivity clamp, and clamp loader complex. The holC gene encodes an accessory protein (known as χ) 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, we isolate spontaneous suppressor mutants in a ΔholC 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 alleles of sspA, encoding 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 (α), RpoB (ß), and RpoC (ß') RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. 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 An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of Escherichia 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 avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC's absence.

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 dcmΔ 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 and independent of double-strand break repair. The SOS response helps ameliorate the mutagenic effect of 5-azaC but this is not a result of SOS-induced DNA polymerases since their induction, especially PolIV, seems to stimulate QP-associated mutagenesis. Cell division regulator SulA was also required for recovery of QP mutants induced by 5-azaC. In the absence of Lon protease, Dcm-dependent QP-mutagenesis is strongly elevated, suggesting it may play a role in DPC 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.

Alternative complexes formed by the Escherichia coli clamp loader accessory protein HolC (x) with replication protein HolD (ψ) and repair protein YoaA.

Efficient and faithful replication of DNA is essential for all organisms. However, the replication fork frequently encounters barriers that need to be overcome to ensure cell survival and genetic stability. Cells must carefully balance and regulate replication vs. repair reactions. In Escherichia coli, the replisome consists of the DNA polymerase III holoenzyme, including DNA polymerase, proofreading exonuclease, processivity clamp and clamp loader, as well as a fork helicase, DnaB and primase, DnaG. We provide evidence here that one component of the clamp loader complex, HolC (or χ) plays a dual role via its ability to form 2 mutually exclusive complexes: one with HolD (or ψ) that recruits the clamp-loader and hence the DNA polymerase holoenzyme and another with helicase-like YoaA protein, a DNA-damage inducible repair protein. By yeast 2 hybrid analysis, we show that two residues of HolC, F64 and W57, at the interface in the structure with HolD, are required for interaction with HolD and for interaction with YoaA. Mutation of these residues does not interfere with HolC's interaction with single-strand DNA binding protein, SSB. In vivo, these mutations fail to complement the poor growth and sensitivity to azidothymidine, a chain-terminating replication inhibitor. In support of the notion that these are exclusive complexes, co-expression of HolC, HolD and YoaA, followed by pulldown of YoaA, yields a complex with HolC but not HolD. YoaA fails to pulldown HolC-F64A. We hypothesize that HolC, by binding with SSB, can recruit the DNA polymerase III holoenzyme through HolD, or an alternative repair complex with YoaA helicase.