Biomedical subjects

H Bierne

Publications and source records attributed to H Bierne.

5 recordsLinked to original sources

PubMed1995

Competition between parental and recombinant plasmids affects the measure of recombination frequencies.

Recombination frequencies in multicopy plasmids are generally deduced from the rate of appearance of cells expressing a recombinant phenotype (i.e., "recombinant cells"). Detection of these cells requires not only the formation of a recombinant molecule but also the establishment of this molecule in the presence of the resident incompatible parental plasmid. Differences in fitness between parental and recombinant molecules will affect this establishment and could have great consequences for plasmid recombination measurements. To test this hypothesis, we compared recombination frequencies when the recombinant plasmid has or does not have a replication advantage over the parental plasmid. We used pBR322-derived plasmids which carry or lack the replication terminator TerB; recombination took place between directly repeated sequences of 16 bp and deleted TerB from the plasmid. The rate of appearance of recombinant cells strongly increased when the Tus/Ter system was active; however, we found no evidence for direct stimulation of recombination between direct repeats by replication fork stalling. The main factor responsible for the increase in the rate of appearance of recombinant cells when the parental plasmid carries TerB is the facilitated establishment of the recombinant plasmid since: (i) the transformation efficiency of the recombinant plasmid is higher in cells containing the Ter+ parent than in cells containing the Ter- parent; (ii) most recombinant plasmids did not lead to the appearance of recombinant cells when pBR322 was was not blocked by Tus, whereas the presence of TerB allows the detection of most events; and (iii) decreasing the parental plasmid copy number without modification of the recombinant plasmid leads to an exponential increase in the rate of appearance of recombinant cells. Our results show that the level of competition between parent and recombinant plasmids can greatly affect plasmid recombination frequencies deduced from the measure of recombinant cells. This effect can be as high as several orders of magnitude.

Base Sequence↗

PubMed1994

When replication forks stop.

DNA synthesis is an accurate and very processive phenomenon, yet chromosome replication does not proceed at a constant rate and progression of the replication fork can be impeded. Several structural and functional features of the template can modulate the rate of progress of the replication fork. These include DNA secondary structures, DNA damage and occupied protein-binding sites. In addition, prokaryotes contain sites where replication is specifically arrested. DNA regions at which the replication machinery is blocked or transiently slowed could be particularly susceptible to genome rearrangements. Illegitimate recombination, a ubiquitous phenomenon which may have dramatic consequences, occurs by a variety of mechanisms. The observation that some rearrangements might be facilitated by a pause in replication could provide a clue in elucidating these processes. In support of this, some homologous and illegitimate recombination events have already been correlated with replication pauses or arrest sites.

Chromosomes↗

PubMed1994

Flanking sequences affect replication arrest at the Escherichia coli terminator TerB in vivo.

We have analyzed the effect of flanking sequences on Tus-induced replication arrest. pBR322 plasmid derivatives which carry the Escherichia coli replication terminator TerB at different locations were used. Efficiency of the replication arrest was estimated from the plasmid copy number and transformation frequency of tus+ cells. We found that flanking sequences do affect replication arrest efficiency, a weak arrest being correlated with the presence of an AT-rich region which is replicated just before TerB. Some sequences located after the replication terminator can also affect replication termination. We propose that the AT-rich regions might impair binding of the Tus protein to the TerB sequence or facilitate helicase-induced unwinding of DNA and Tus displacement from the TerB site.

Bacterial Proteins↗

PubMed1993

Mechanisms of illegitimate recombination.

Illegitimate recombination, which is one of the major causes of genome rearrangements, can occur in a number of ways. These might involve enzymes which cut and join DNA or enzymes which replicate DNA, as illustrated by two examples: (i) formation of deletions at the replication origin (ori) of an Escherichia coli bacteriophage, M13; and (ii) excision of E. coli transposon Tn10. It is proposed that a common theme to various ways by which illegitimate recombination can occur might be the capacity to create ends in the DNA molecule and to make the ends meet.

Bacteriophage M13↗

PubMed1991

The replication termination signal terB of the Escherichia coli chromosome is a deletion hot spot.

Hybrids composed of phage M13, plasmid pBR322 and the termination signal of Escherichia coli chromosome replication terB were used to show that arrest of DNA synthesis creates a very efficient deletion hot spot. Up to 80% of deletions occurring in these hybrids had one deletion end-point at terB provided that (i) terB was oriented to arrest M13 and pBR322 leading strand synthesis; and (ii) the host cells contained the Tus protein necessary for arresting DNA synthesis at terB. The position of terB and the flanking sequences had little effect on deletion hot spot activity. About 90% of the deletions at terB ended 5-6 nucleotides in front of the major replication arrest site. We propose two models to account for deletion formation and speculate that many genome rearrangements may be due to the pausing of DNA replication.

Base Sequence↗