Investigating Nucleotide Excision DNA Repair by Single-Molecule Imaging of Quantum Dot Labeled Proteins Reveals Unique Scanning Mechanisms (original) (raw)
Related papers
The EMBO Journal, 1999
The parA locus of plasmid R1 encodes a prokaryotic centromere-like system that mediates genetic stabilization of plasmids by an unknown mechanism. The locus codes for two proteins, ParM and ParR, and a centromere-like DNA region (parC) to which the ParR protein binds. We showed recently that ParR mediates specific pairing of parC-containing DNA molecules in vitro. To obtain further insight into the mechanism of plasmid stabilization, we examined the intracellular localization of the components of the parA system. We found that ParM forms discrete foci that localize to specific cellular regions in a simple, yet dynamic pattern. In newborn cells, ParM foci were present close to both cell poles. Concomitant with cell growth, new foci formed at mid-cell. A point mutation that abolished the ATPase activity of ParM simultaneously prevented cellular localization and plasmid partitioning. A parAcontaining plasmid localized to similar sites, i.e. close to the poles and at mid-cell, thus indicating that the plasmid co-localizes with ParM. Double labelling of single cells showed that plasmid DNA and ParM indeed co-localize. Thus, our data indicate that parA is a true partitioning system that mediates pairing of plasmids at mid-cell and subsequently moves them to the cell poles before cell division.
Mechanism of DNA segregation in prokaryotes: Replicon pairing by parC of plasmid R1
Proceedings of the National Academy of Sciences, 1998
Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division. The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation. The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC. The ParR protein binds to parC in vivo and in vitro. The ParM protein is an ATPase that interacts with ParR specifically bound to parC. Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules. The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase. The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM. Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments. The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity. Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro. These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes. MATERIALS AND METHODS Plasmids. Plasmid pMD330 (11) contains as the only R1 derived sequence the minimal parC region cloned into pUC19 (34). Plasmid pAB1922 contains parC22, in which the spacer region of 39 bp between the two sets of direct repeats has been deleted (13) and pMD333 contains parC33, in which the five downstream repeats of parC have been deleted (11). Electron Microscopy Analysis. Supercoiled or SspI digested pMD330 DNA (40-500 ng) was incubated with ParR (25-500 ng), ParM (25-500 ng), ParM D170E (100-250 ng), or E. coli RNA polymerase (40-250 ng) for 15 min at 37°C in 20 l of 30 mM triethanolamine⅐HCl (pH 7.5), 50 mM KCl, 5 mM MgCl 2 , 1 mM DTT, and, when appropriate, 1 mM ATP, ADP, adenylylimidophosphat, or adenosine-5Ј-O-(3-thiotriphosphate). The complexes were subsequently fixed with 0.2% glutaraldehyde for 15 min at 37°C and the glutaraldehyde was removed by using Micro Bio-Spin 30 gel filtration columns (Bio-Rad). After cleavage of the DNA with SspI, the gel filtration step was repeated. Adsorption to mica, rotational shadowing with platinum, and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
A conserved mechanism drives partition complex assembly on bacterial chromosomes and plasmids
Molecular Systems Biology
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A Nucleotide Switch in the Escherichia coli DnaA Protein Initiates Chromosomal Replication
Journal of Biological Chemistry, 2002
The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp-and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.
Chromosome segregation: pushing plasmids apart
Current biology : CB, 2002
The ParM ATPase from Escherichia coli plasmid R1 assembles into F-actin-like filaments which appear to push replicated copies of the plasmid to opposite ends of the cell, ensuring partitioning into daughter cells. Might bacterial chromosomes use a similar mitotic strategy for segregation?
Chromosome segregation by the Escherichia coli Min system
Molecular Systems Biology, 2013
The mechanisms underlying chromosome segregation in prokaryotes remain a subject of debate and no unifying view has yet emerged. Given that the initial disentanglement of duplicated chromosomes could be achieved by purely entropic forces, even the requirement of an active prokaryotic segregation machinery has been questioned. Using computer simulations, we show that entropic forces alone are not sufficient to achieve and maintain full separation of chromosomes. This is, however, possible by assuming repeated binding of chromosomes along a gradient of membrane-associated tethering sites toward the poles. We propose that, in Escherichia coli, such a gradient of membrane tethering sites may be provided by the oscillatory Min system, otherwise known for its role in selecting the cell division site. Consistent with this hypothesis, we demonstrate that MinD binds to DNA and tethers it to the membrane in an ATP-dependent manner. Taken together, our combined theoretical and experimental results suggest the existence of a novel mechanism of chromosome segregation based on the Min system, further highlighting the importance of active segregation of chromosomes in prokaryotic cell biology.
Nature Communications
The efficient segregation of replicated genetic material is an essential step for cell division. Bacterial cells use several evolutionarily-distinct genome segregation systems, the most common of which is the type I Par system. It consists of an adapter protein, ParB, that binds to the DNA cargo via interaction with the parS DNA sequence; and an ATPase, ParA, that binds nonspecific DNA and mediates cargo transport. However, the molecular details of how this system functions are not well understood. Here, we report the cryo-EM structure of the Vibrio cholerae ParA2 filament bound to DNA, as well as the crystal structures of this protein in various nucleotide states. These structures show that ParA forms a left-handed filament on DNA, stabilized by nucleotide binding, and that ParA undergoes profound structural rearrangements upon DNA binding and filament assembly. Collectively, our data suggest the structural basis for ParA’s cooperative binding to DNA and the formation of high ParA ...
Asymmetric chromosome segregation and cell division in DNA damage-induced bacterial filaments
2020
Faithful propagation of life requires coordination of DNA replication and segregation with cell growth and division. In bacteria, this results in cell size homeostasis and periodicity in replication and division. The situation is perturbed under stress such as DNA damage, which induces filamentation as cell cycle progression is blocked to allow for repair. Mechanisms that release this morphological state for re-entry into wild type growth are unclear. Here we show that damage recovery is mediated via asymmetric division of Escherichia coli filaments, producing short daughter cells with wild type size and growth dynamics. Division restoration at this polar site is governed by coordinated action of divisome positioning by the Min system and modulation of division licensing by the terminus region of the chromosome, with MatP playing a central role in this process. Collectively, our study highlights a key role for concurrency between chromosome (and specifically terminus) segregation an...