Biomedical subjects

Therese Brendler

Publications and source records attributed to Therese Brendler.

5 recordsLinked to original sources

PubMed2005

Crystal structure of a SeqA-N filament: implications for DNA replication and chromosome organization.

Escherichia coli SeqA binds clusters of transiently hemimethylated GATC sequences and sequesters the origin of replication, oriC, from methylation and premature reinitiation. Besides oriC, SeqA binds and organizes newly synthesized DNA at replication forks. Binding to multiple GATC sites is crucial for the formation of stable SeqA-DNA complexes. Here we report the crystal structure of the oligomerization domain of SeqA (SeqA-N). The structural unit of SeqA-N is a dimer, which oligomerizes to form a filament. Mutations that disrupt filament formation lead to asynchronous DNA replication, but the resulting SeqA dimer can still bind two GATC sites separated from 5 to 34 base pairs. Truncation of the linker between the oligomerization and DNA-binding domains restricts SeqA to bind two GATC sites separated by one or two full turns. We propose a model of a SeqA filament interacting with multiple GATC sites that accounts for both origin sequestration and chromosome organization.

Bacterial Outer Membrane Proteins↗

PubMed2005

Hda inactivation of DnaA is the predominant mechanism preventing hyperinitiation of Escherichia coli DNA replication.

Initiation of DNA replication from the Escherichia coli chromosomal origin is highly regulated, assuring that replication occurs precisely once per cell cycle. Three mechanisms for regulation of replication initiation have been proposed: titration of free DnaA initiator protein by the datA locus, sequestration of newly replicated origins by SeqA protein and regulatory inactivation of DnaA (RIDA), in which active ATP-DnaA is converted to the inactive ADP-bound form. DNA microarray analyses showed that the level of initiation in rapidly growing cells that lack datA was indistinguishable from that in wild-type cells, and that the absence of SeqA protein caused only a modest increase in initiation, in agreement with flow-cytometry data. In contrast, cells lacking Hda overinitiated replication twofold, implicating RIDA as the predominant mechanism preventing extra initiation events in a cell cycle.

Adenosine Triphosphatases↗

PubMed2005

Surprising dependence on postsegregational killing of host cells for maintenance of the large virulence plasmid of Shigella flexneri.

Low-copy-number plasmids all encode multiple systems to ensure their propagation, including replication, partition (active segregation), and postsegregational killing (PSK) systems. PSK systems kill those rare cells that lose the plasmid due to replication or segregation errors. PSK systems should not be used as the principle means of maintaining the plasmid. The metabolic cost of killing the many cured cells that would arise from random plasmid segregation is far too high. Here we describe an interesting exception to this rule. Maintenance of the large virulence plasmid of Shigella flexneri is highly dependent on one of its PSK systems, mvp, at 37 degrees C, the temperature experienced during pathogenesis. At 37 degrees C, the plasmid is very unstable and mvp efficiently kills the resulting cured bacterial cells. This imposes a major growth disadvantage on the virulent bacterial population. The systems that normally ensure accurate plasmid replication and segregation are attenuated or overridden at 37 degrees C. At 30 degrees C, a temperature encountered by Shigella in the outside environment, the maintenance systems function normally and the plasmid is no longer dependent on mvp. We discuss why the virulent pathogen tolerates this self-destructive method of propagation at the temperature of infection.

Cell Division↗

PubMed2004

Interplay between plasmid partition and postsegregational killing systems.

Active partition systems and postsegregational killing (PSK) systems are present together in naturally occurring low-copy-number plasmids. Theory suggests that PSK may act as the ultimate determinant of plasmid retention, whereas the partition system may minimize the growth penalty to the host, resulting in a near-ideal symbiosis when the systems combine. Here, we prove the validity of this principle for a specific case involving the P1par system and the mvp PSK system.

Cell Division↗

PubMed2002

Transcriptional interference by a complex formed at the centromere-like partition site of plasmid P1.

The partition site, parS, promotes accurate segregation of the replicated P1 plasmid to daughter cells when the P1-encoded ParA and ParB proteins are supplied. The parS site was inserted into the Escherichia coli chromosome between the promoter and the structural gene for beta-galactosidase, lacZ. There was little interference with lacZ expression when ParA and ParB were supplied in trans. However, when a mutant ParA protein, ParAM314I, was supplied along with ParB, expression of lacZ was shut down. ParAM314I, ParB, and parS appear to form a nucleoprotein complex that blocks transcription. Mutations in parA and parB that relieved the parAM314I-dependent block were found. In addition, new mutations which impose the block were selected. Five of the latter mapped to parA and one to parB; all had a propagation-defective phenotype (Par(PD)) similar to that of parAM314I. Thus, whereas a null par mutant P1 plasmid segregates its DNA randomly, these mutants prevent even random distribution of the plasmid. We propose that ParA protein normally interacts transiently with the ParB-parS complex for partition to proceed but that the mutations block ParA dissociation. This "permanent" ParA-ParB-parS complex acts as a transcription block. Consistent with this hypothesis, we found that three of the seven blocking mutations lie within regions of ParA and ParB that are known to interact with each other. When the transcription block is imposed, regional silencing of nearby genes occurs. However, the requirement for ParA and a mutant parA or parB allele distinguishes the transcription block from the regional ParB-dependent gene silencing previously described.

Bacterial Proteins↗