In Escherichia coli, several enzymes have been identified that participate in completing replication on the chromosome, including RecG, SbcCD, ExoI, and RecBCD. However, other enzymes are likely to be involved and the precise enzymatic mechanism by which this reaction occurs remains unknown. Two steps predicted to be necessary to complete replication are removal of Okazaki RNA fragments and ligation of the nascent strands at convergent replication forks. E. coli encodes two RNases that remove RNA-DNA hybrids, rnhA and rnhB, as well as two ligases, ligA and ligB. Here, we used replication profiling to show that rnhA and ligA, encoding RNase HI and Ligase A, participate in the completion reaction. Deletion of rnhA impaired the ability to complete replication and resulted in over-replication in the terminus region. It additionally suppressed initiation events from oriC, suggesting a role for the enzyme in oriC-dependent initiation, as has been suggested previously. We also show that a temperature-sensitive mutation in Ligase A led to over-replication at sites where replication completes, and that degradation at these sites occurred upon shifting to the nonpermissive temperature. Deletion of rnhB or ligB did not affect the growth or profile of replication on the genome.
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UV‐induced DNA damage disrupts the coordination between replication initiation, elongation and completion
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
- Award ID(s):
- 1916625
- NSF-PAR ID:
- 10453446
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Genes to Cells
- Volume:
- 26
- Issue:
- 2
- ISSN:
- 1356-9597
- Page Range / eLocation ID:
- p. 94-108
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Key points Heart failure (HF), the leading cause of death in developed countries, occurs in the setting of reduced (HFrEF) or preserved (HFpEF) ejection fraction. Unlike HFrEF, there are no effective treatments for HFpEF, which accounts for ∼50% of heart failure.
Abnormal intracellular calcium dynamics in cardiomyocytes have major implications for contractility and rhythm, but compared to HFrEF, very little is known about calcium cycling in HFpEF.
We used rat models of HFpEF and HFrEF to reveal distinct differences in intracellular calcium regulation and excitation‐contraction (EC) coupling.
While HFrEF is characterized by defective EC coupling at baseline, HFpEF exhibits enhanced coupling fidelity, further aggravated by a reduction in β‐adrenergic sensitivity.
These differences in EC coupling and β‐adrenergic sensitivity may help explain why therapies that work in HFrEF are ineffective in HFpEF.
Abstract Heart failure with reduced or preserved ejection fraction (respectively, HFrEF and HFpEF) is the leading cause of death in developed countries. Although numerous therapies improve outcomes in HFrEF, there are no effective treatments for HFpEF. We studied phenotypically verified rat models of HFrEF and HFpEF to compare excitation‐contraction (EC) coupling and protein expression in these two forms of heart failure. Dahl salt‐sensitive rats were fed a high‐salt diet (8% NaCl) from 7 weeks of age to induce HFpEF. Impaired diastolic relaxation and preserved ejection fraction were confirmed in each animal echocardiographically, and clinical signs of heart failure were documented. To generate HFrEF, Sprague‐Dawley (SD) rats underwent permanent left anterior descending coronary artery ligation which, 8–10 weeks later, led to systolic dysfunction (verified echocardiographically) and clinical signs of heart failure. Calcium (Ca2+) transients were measured in isolated cardiomyocytes under field stimulation or patch clamp. Ultra‐high‐speed laser scanning confocal imaging captured Ca2+sparks evoked by voltage steps. Western blotting and PCR were used to assay changes in EC coupling protein and RNA expression. Cardiomyocytes from rats with HFrEF exhibited impaired EC coupling, including decreased Ca2+transient (CaT) amplitude and defective couplon recruitment, associated with transverse (t)‐tubule disruption. In stark contrast, HFpEF cardiomyocytes showed saturated EC coupling (increased
I Ca, high probability of couplon recruitment with greater Ca2+release synchrony, increased CaT) and preserved t‐tubule integrity. β‐Adrenergic stimulation of HFpEF myocytes with isoprenaline (isoproterenol) failed to elicit robust increases inI Caor CaT and relaxation kinetics. Fundamental differences in EC coupling distinguish HFrEF from HFpEF. -
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Abstract Telomeres cap the physical ends of eukaryotic chromosomes to ensure complete DNA replication and genome stability. Heritable natural variation in telomere length exists in yeast, mice, plants and humans at birth; however, major effect loci underlying such polymorphism remain elusive. Here, we employ quantitative trait locus (QTL) mapping and transgenic manipulations to identify genes controlling telomere length set point in a multi-parent
Arabidopsis thaliana mapping population. We detect several QTL explaining 63.7% of the total telomere length variation in the Arabidopsis MAGIC population. Loss-of-function mutants of theNOP2A candidate gene located inside the largest effect QTL and of two other ribosomal genesRPL5A andRPL5B establish a shorter telomere length set point than wild type. These findings indicate that evolutionarily conserved components of ribosome biogenesis and cell proliferation pathways promote telomere elongation. -
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Abstract A peptidoglycan (PG) cell wall composed of glycans crosslinked by short peptides surrounds most bacteria and protects them against osmotic rupture. In
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Summary Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca2+]cyt) increase as a second messenger. The downstream PM Ca2+channels remain enigmatic. Here, the
Arabidopsis thaliana Ca2+channel subunit CYCLIC NUCLEOTIDE‐GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+]cytsignalling in roots.Extracellular ATP‐induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+]cytwere measured with aequorin, and root transcriptional changes were determined by quantitative real‐time PCR. Two
cngc2 loss‐of‐function mutants were used:cngc2‐3 anddefence not death1 (which expresses cytosolic aequorin).Extracellular ATP‐induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+dependent, requiring CNGC2 but not CNGC4 (its channel co‐subunit in immunity signalling). Activation of PM Ca2+influx currents also required CNGC2. The eATP‐induced [Ca2+]cytincrease and transcriptional response in
cngc2 roots were significantly impaired.CYCLIC NUCLEOTIDE‐GATED CHANNEL2 is required for eATP‐induced epidermal Ca2+influx, causing depolarization leading to [Ca2+]cytincrease and damage‐related transcriptional response.
