- Award ID(s):
- 1916625
- NSF-PAR ID:
- 10274577
- Editor(s):
- Rhind, N
- Date Published:
- Journal Name:
- G3 Genes|Genomes|Genetics
- Volume:
- 11
- Issue:
- 4
- ISSN:
- 2160-1836
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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ABSTRACT Divalent metals such as iron and manganese play an important role in the cellular response to oxidative challenges and are required as cofactors by many enzymes. However, how these metals affect replication after oxidative challenge is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. We show that the manganese-dependent recovery of DNA synthesis occurs independent of lesion repair, modestly improves cell survival, and is associated with elevated rates of mutagenesis. The Mn-dependent mutagenesis involves both replicative and translesion polymerases and requires prior disruption by H 2 O 2 to occur. Taking these findings together, we propose that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. The data suggest that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity. IMPORTANCE Iron and manganese play important roles in how cell’s cope with oxygen stress. However, how these metals affect the ability of cells to replicate after oxidative challenges is not known. Here, we show that replication in Escherichia coli is inhibited following a challenge with hydrogen peroxide and requires manganese for the rapid recovery of DNA synthesis. The manganese-dependent recovery of DNA synthesis occurs independently of lesion repair and modestly improves survival, but it also increases the mutation rate in cells. The results imply that replication in E. coli is likely to utilize an iron-dependent enzyme(s) that becomes oxidized and inactivated during oxidative challenges. We propose that manganese remetallates these or alternative enzymes to allow genomic DNA replication to resume, although with reduced fidelity.more » « less
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Following hydrogen peroxide treatment, ferrous iron (Fe2+) is oxidized to its ferric form (Fe3+), stripping it from and inactivating iron-containing proteins. Many mononuclear iron enzymes can be remetallated by manganese to restore function, while other enzymes specifically utilize manganese as a cofactor, having redundant activities that compensate for iron-depleted counterparts. DNA replication relies on one or more iron-dependent protein(s) as synthesis abates in the presence of hydrogen peroxide and requires manganese in the medium to resume. Here, we show that manganese transporters regulate the ability to resume replication following oxidative challenge in Escherichia coli. The absence of the primary manganese importer, MntH, impairs the ability to resume replication; whereas deleting the manganese exporter, MntP, or transporter regulator, MntR, dramatically increases the rate of recovery. Unregulated manganese import promoted recovery even in the absence of Fur, which maintains iron homeostasis. Similarly, replication was not restored in oxyR mutants, which cannot upregulate manganese import following hydrogen peroxide stress. Taken together, the results define a central role for manganese transport in restoring replication following oxidative stress.more » « less
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Abstract Replication initiation, elongation and completion are tightly coordinated to ensure that all sequences replicate precisely once each generation. UV‐induced DNA damage disrupts replication and delays elongation, which may compromise this coordination leading to genome instability and cell death. Here, we profiled the
Escherichia coli genome as it recovers from UV irradiation to determine how these replicational processes respond. We show thatoriC initiations continue to occur, leading to copy number enrichments in this region. At late times, the combination of neworiC initiations and delayed elongating forks converging in the terminus appear to stress or impair the completion reaction, leading to a transient over‐replication in this region of the chromosome. In mutants impaired for restoring elongation, includingrecA ,recF anduvrA , the genome degrades or remains static, suggesting that cell death occurs early after replication is disrupted, leaving partially duplicated genomes. In mutants impaired for completing replication, includingrecBC ,sbcCD xonA andrecG , the recovery of elongation and initiation leads to a bottleneck, where the nonterminus region of the genome is amplified and accumulates, indicating that a delayed cell death occurs in these mutants, likely resulting from mis‐segregation of unbalanced or unresolved chromosomes when cells divide. -
PREMISE Nucleic acid integrity can be compromised under many abiotic stresses. To date, however, few studies have considered whether nucleic acid damage and damage repair play a role in cold‐stress adaptation. A further insufficiently explored question concerns how age affects cold stress adaptation among mature perennials. As a plant ages, the optimal trade‐off between growth and stress tolerance may shift.
METHODS Oxidative damage to RNA and expression of genes involved in DNA repair were compared in multiple mature cohorts of
Thinopyrum intermedium (an emerging perennial cereal) and in wheat and barley under intermittent freezing stress and under nonfreezing conditions. Activity of glutathione peroxidase (GPX) and four other antioxidative enzymes was also measured under these conditions. DNA repair genes included photolyases involved in repairing ultraviolet‐induced damage and two genes involved in repairing oxidatively induced damage (ERCC1, RAD23 ).RESULTS Freezing stress was accompanied by large increases in photolyase expression and
ERCC1 expression (in wheat andThinopyrum ) and in GPX and GR activity (particularly inThinopyrum ). This is the first report of DNA photolyases being overexpressed under freezing stress. OlderThinopyrum had lower photolyase expression and less freezing‐induced overexpression ofERCC1 . YoungerThinopyrum plants sustained more oxidative damage to RNA.CONCLUSIONS Overexpression of DNA repair genes is an important aspect of cold acclimation. When comparing adult cohorts, aging was associated with changes in the freezing stress response, but not with overall increases or decreases in stress tolerance.
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Abstract The Msh2–Msh3 mismatch repair (MMR) complex in Saccharomyces cerevisiae recognizes and directs repair of insertion/deletion loops (IDLs) up to ∼17 nucleotides. Msh2–Msh3 also recognizes and binds distinct looped and branched DNA structures with varying affinities, thereby contributing to genome stability outside post-replicative MMR through homologous recombination, double-strand break repair (DSBR) and the DNA damage response. In contrast, Msh2–Msh3 promotes genome instability through trinucleotide repeat (TNR) expansions, presumably by binding structures that form from single-stranded (ss) TNR sequences. We previously demonstrated that Msh2–Msh3 binding to 5′ ssDNA flap structures interfered with Rad27 (Fen1 in humans)-mediated Okazaki fragment maturation (OFM) in vitro. Here we demonstrate that elevated Msh2–Msh3 levels interfere with DNA replication and base excision repair in vivo. Elevated Msh2–Msh3 also induced a cell cycle arrest that was dependent on RAD9 and ELG1 and led to PCNA modification. These phenotypes also required Msh2–Msh3 ATPase activity and downstream MMR proteins, indicating an active mechanism that is not simply a result of Msh2–Msh3 DNA-binding activity. This study provides new mechanistic details regarding how excess Msh2–Msh3 can disrupt DNA replication and repair and highlights the role of Msh2–Msh3 protein abundance in Msh2–Msh3-mediated genomic instability.