Epistatic Roles for Pseudomonas aeruginosa MutS and DinB (DNA Pol IV) in Coping with Reactive Oxygen Species-Induced DNA Damage

Pseudomonas aeruginosa is especially adept at colonizing the airways of individuals afflicted with the autosomal recessive disease cystic fibrosis (CF). CF patients suffer from chronic airway inflammation, which contributes to lung deterioration. Once established in the airways, P. aeruginosa continuously adapts to the changing environment, in part through acquisition of beneficial mutations via a process termed pathoadaptation. MutS and DinB are proposed to play opposing roles in P. aeruginosa pathoadaptation: MutS acts in replication-coupled mismatch repair, which acts to limit spontaneous mutations; in contrast, DinB (DNA polymerase IV) catalyzes error-prone bypass of DNA lesions, contributing to mutations. As part of an ongoing effort to understand mechanisms underlying P. aeruginosa pathoadaptation, we characterized hydrogen peroxide (H2O2)-induced phenotypes of isogenic P. aeruginosa strains bearing different combinations of mutS and dinB alleles. Our results demonstrate an unexpected epistatic relationship between mutS and dinB with respect to H2O2-induced cell killing involving error-prone repair and/or tolerance of oxidized DNA lesions. In striking contrast to these error-prone roles, both MutS and DinB played largely accurate roles in coping with DNA lesions induced by ultraviolet light, mitomycin C, or 4-nitroquinilone 1-oxide. Models discussing roles for MutS and DinB functionality in DNA damage-induced mutagenesis, particularly during CF airway colonization and subsequent P. aeruginosa pathoadaptation are discussed

Breast cancer management pathways during the COVID-19 pandemic: outcomes from the UK ‘Alert Level 4’ phase of the B-MaP-C study

Abstract: Background: The B-MaP-C study aimed to determine alterations to breast cancer (BC) management during the peak transmission period of the UK COVID-19 pandemic and the potential impact of these treatment decisions. Methods: This was a national cohort study of patients with early BC undergoing multidisciplinary team (MDT)-guided treatment recommendations during the pandemic, designated ‘standard’ or ‘COVID-altered’, in the preoperative, operative and post-operative setting. Findings: Of 3776 patients (from 64 UK units) in the study, 2246 (59%) had ‘COVID-altered’ management. ‘Bridging’ endocrine therapy was used (n = 951) where theatre capacity was reduced. There was increasing access to COVID-19 low-risk theatres during the study period (59%). In line with national guidance, immediate breast reconstruction was avoided (n = 299). Where adjuvant chemotherapy was omitted (n = 81), the median benefit was only 3% (IQR 2–9%) using ‘NHS Predict’. There was the rapid adoption of new evidence-based hypofractionated radiotherapy (n = 781, from 46 units). Only 14 patients (1%) tested positive for SARS-CoV-2 during their treatment journey. Conclusions: The majority of ‘COVID-altered’ management decisions were largely in line with pre-COVID evidence-based guidelines, implying that breast cancer survival outcomes are unlikely to be negatively impacted by the pandemic. However, in this study, the potential impact of delays to BC presentation or diagnosis remains unknown

<i>P. aeruginosa</i> DinB catalyzes both accurate and error-prone bypass of 8-oxo-dG <i>in vitro</i>.^a

<p><i><b>^a</b></i>Bypass activity was measured <i>in vitro</i> using a synthetic 13-mer oligonucleotide primer (5′-TGG CAG CCG GTC A-3′) annealed to a synthetic 20-mer template strand bearing either dG or 8-oxo-dG (3′-ACC GTC GGC CAG T<b><i>x</i></b>C CCA AA-5′, where <b><i>x</i></b> represents either dG or 8-oxo-dG).</p><p><i><b>^b</b></i>Neither dTTP nor dGTP were incorporated opposite template dG at a detectable level.</p><p><i><b>^c</b></i>Values shown represent the average of at least 3 independent experiments, ± the standard deviation.</p><p><i><b>^d</b></i>Incorporation of dATP opposite template dG was not detected (<i>nd</i>).</p><p><i><b>^e</b></i>Not applicable (<i>na</i>).</p

Contribution of <i>dinB</i> and/or <i>mutS</i> function to H₂O₂-induced mutagenesis.

<p>H₂O₂-induced mutagenesis was measured in <i>mutS⁺</i> (<b>A</b>), or <i>mutS</i>::IS<i>phoA</i>/hah (<b>B</b>) <i>dinB⁺</i> and Δ<i>dinB</i>::<i>aacC1</i> strains, bearing the indicated plasmids. Strains examined include MPAO1 (wild-type) bearing pUCP20T, WFPA334 (Δ<i>dinB</i>::<i>aacC1</i>) bearing pUCP20T (control) or pAR101 (<i>dinB⁺</i>), MPA32417 (<i>mutS</i>::IS<i>phoA</i>/hah) bearing pUCP20T (control), or UBPA100 bearing pUCP20T (control) or pAR101 (<i>dinB⁺</i>). H₂O₂-induced mutation frequencies represent the average of 4–6 independent experiments. Error bars represent the standard deviation. <i>P</i>-values are indicated, and were calculated using the Student's <i>t</i>-test.</p

Contributions of <i>dinB</i> and/or <i>mutS</i> function to survival following exposure to ROS.

<p>Respective H₂O₂ sensitivities (<b>A</b>), and catalase activities for cell free extracts (<b>B</b>) of isogenic <i>P. aeruginosa</i> strains MPAO1 (wild-type) bearing pUCP20T, WFPA334 (Δ<i>dinB</i>::<i>aacC1</i>) bearing pUCP20T (control) or pAR101 (<i>dinB⁺</i>), MPA32417 (<i>mutS</i>::IS<i>phoA</i>/hah) bearing pUCP20T (control) or p<i>mutS</i> (<i>mutS⁺</i>), and UBPA100 (Δ<i>dinB</i>::<i>aacC1 mutS</i>::IS<i>phoA</i>/hah) bearing pUCP20T were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018824#s4" target="_blank"><i>Materials and Methods</i></a>. H₂O₂ sensitivities represent the average of 4 independent experiments, while catalase activities represent the average of 3 independent experiments; error bars represent the standard deviation. Based on a two-way ANOVA with Bonferroni post-test, there was a significant interaction between strain and concentration of H₂O₂ in (<b>A</b>), and differences in H₂O₂ sensitivity of wild-type and <i>mutS</i> and <i>dinB</i> strains were statistically significant (<i>p</i><0.05). Based on a one-way ANOVA with Dunnett's post-test, differences in catalase activity in (<b>B</b>) were not significant (<i>p</i>>0.05).</p

Nucleotide sequence analysis of <i>rpoB</i> alleles recovered from spontaneous or H₂O₂-induced Rif^R<i>P. aeruginosa</i> strains.^a

<p><i><b>^a</b></i>Spontaneous and H₂O₂-induced (25 mM H₂O₂) mutations were identified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018824#s4" target="_blank"><i>Materials and Methods</i></a>. The region of <i>rpoB</i> encompassing amino acids 499–582 of the β subunit of RNA polymerase was PCR amplified from 18–20 independent Rif^R clones for each strain and subjected to automated nucleotide sequence analysis.</p><p><i><b>^b</b></i>Each nucleotide substitution (underlined) is shown in the context of its respective codon.</p><p><i><b>^c</b></i>The number of times that each mutation was identified in the group of 18–20 that was sequenced is indicated; <i>nd</i>, the indicated mutation was not detected.</p><p><i><b>^d</b></i>Seven of the 19 Rif^R clones examined did not contain a mutation within amino acids 499–582.</p><p><i><b>^e</b></i>One of the 19 Rif^R clones examined did not contain a mutation within amino acids 499–582.</p><p><i><b>^f</b></i>These mutations were present in the same <i>rpoB</i> allele.</p><p><i><b>^g</b></i>Three of the 20 Rif^R clones examined did not contain a mutation within amino acids 499–582.</p><p><i><b>^h</b></i>Two of the 20 Rif^R clones examined did not contain a mutation within amino acids 499–582.</p

Summary of spontaneous and H₂O₂-induced base substitutions in <i>rpoB</i> that confer Rif^R.

<p>Results of <i>rpoB</i> DNA sequence analysis for spontaneous (<b>A</b>) and H₂O₂-induced (<b>B</b>) Rif^R<i>P. aeruginosa</i> mutants are summarized with respect to the types of nucleotide substitution observed. Frequency refers to the occurrence of each observed base substitution mutation as a function of the total number of spontaneous or H₂O₂-induced Rif^R mutants sequenced for each strain. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018824#pone-0018824-t002" target="_blank">Table 2</a> for details concerning the number of Rif^R clones analyzed for each strain, as well as the specific nucleotide position and substitution of each documented mutation.</p

DinB catalyzes accurate bypass of a model <i>cis-syn</i> thymine cyclobutane dimer <i>in vitro</i>.

<p>Cartoon representation of the 13/20_T = T-mer DNA template (<b>A</b>). T = T represents the <i>cis-syn</i> thymine cyclobutane dimer. Predicted sizes for the starting primer (13-mer), as well as the different possible bypass products (14-mer & 15-mer) are indicated. Representative bypass results for T4 <i>exo^–</i> Pol and <i>P. aeruginosa</i> DinB are shown (<b>B</b>). Positions for the primer (13-mer), and the 14-mer and 15-mer bypass products are indicated. Quantitation of the results of a representative bypass assay obtained with both T4 <i>exo^–</i> Pol and DinB are shown (<b>C</b>).</p

Models to describe roles for <i>P. aeruginosa</i> DinB and MutS in DNA repair, DNA damage tolerance, and DNA damage-induced mutagenesis.

<p>Proposed roles for MutS, DinB, and additional factors (as noted) in coping with ROS-, UV-, and MMC- or 4-NQO–induced (collectively referred to as ‘<i>N²</i>-dG adducts’) DNA lesions are summarized. Fidelities (accurate or error-prone) of MutS and DinB in each pathway (DinB-dependent and DinB-independent) are indicated. See text for additional details.</p

DinB catalyzes accurate bypass of the 5′-dT in a <i>cis-syn</i> thymine cyclobutane dimer <i>in vitro</i>.

<p>Cartoon representation of the 14/20_T = T-mer DNA template (<b>A</b>). T = T represents the <i>cis-syn</i> thymine cyclobutane dimer. Sizes for the starting primer (14-mer), as well as the bypass product (15-mer) are indicated. Bypass efficiency of the 5′-dT of the dimer by DinB as a function of time in the presence of each of the four individual dNTPs is shown (<b>B</b>). Catalytic efficiency with which DinB mediates bypass of the 5′-dT of the dimer in the presence of dATP (<b>C</b>). K_M and k_cat values shown represent the average of at least 3 independent experiments. Error bars represent the standard deviation.</p