Topoisomerases play essential roles in all aspects of DNA metabolism. In all organisms, type II topoisomerases are required for the proper topological separation of newly replicated chromosomal DNA. In E. coli
, Topo IV is the enzyme responsible for accomplishing this task (1
). In its absence, cells arrest with a partition phenotype, characterized by a large nucleoid mass of intertwined chromosomes in the center of a filamentous cell (19
In an attempt to further delineate the flow of information during the terminal stages of DNA replication and cell division, we carried out a genetic screening to detect high-copy suppressors of the temperature-sensitive phenotype of the parE10 allele. This screening yielded dnaX, which encodes the τ and γ subunits of the DNA polymerase III holoenzyme, the replication fork polymerase. Additional characterization of the suppression by dnaX revealed that expression of γ alone, but not expression of τ alone, could almost completely rescue both the temperature-sensitive and partition phenotypes of parE10 E. coli.
However, at this time, we cannot conclude that we have uncovered a distinct role for γ that cannot be accomplished by τ. The expression of τ appears to be toxic to some extent, which could limit the possibility of observing rescue by expression of τ alone. Additionally, rescue of W3110parE10 cells by γ alone was more complete than when τ and γ were expressed together. This increased ability of γ alone to rescue could be the result of either a higher level of expression of γ from the pBR-dnaX-γ construct than from the pBR-dnaX construct, the absence of the toxic effect of τ, or a combination of both factors.
The question of whether γ and τ have distinct roles is an intriguing one that has yet to be answered satisfactorily. Biochemically, τ can substitute for γ, forming a β-loading τ complex (7
), and it has been suggested that only τ, not γ, is required for viability of E. coli
). This is consistent with the observation that γ is not required for replication fork action in vitro during rolling circle DNA replication reconstituted with purified proteins (25
). On the other hand, γ is clearly associated with holoenzyme purified from bulk E. coli
and only γ complex is found free in the cell; τ complex has never been detected (21
How might overproduction of γ result in the observed suppression? There are two obvious possibilities that we have been able to eliminate. (i) Overexpression of γ could cause, either directly or indirectly, overexpression of the ParE10 protein. This might stabilize the polypeptide against denaturation at the nonpermissive temperature. This has been observed, for example, for the ssb-1
temperature-sensitive allele (5
). However, ECL-Western blot analysis has shown that the levels of both ParE and ParC remain constant in W3110parE10
in either the presence or absence of the pBR-dnaX
-γ plasmid at both 32 and 42°C (data not shown). (ii) γ, which is an ATPase that is involved in opening the ring of the β dimer to allow it to encircle the DNA template (33
), might form a novel topoisomerase with either ParC or GyrA that is capable of decatenating the daughter chromosomes. We have assessed this directly by testing whether the purified proteins exhibit such an activity in vitro and have not detected it, even at protein concentrations far in excess of what would be required to observe activity with wild-type Topo IV or DNA gyrase (data not shown).
A third interesting possibility is that overexpression of γ interferes with DNA replication in some way. If so, replication may proceed at a lower rate, thereby reducing the need for rapid unlinking of the replicated duplex DNA and allowing other topoisomerases, such as gyrase, to assist or replace the weakened Topo IV. However, a number of our observations suggest that this is not a likely possibility. If this explanation were true, and DNA replication were slowed to a point where topoisomerases that can decatenate less efficiently than Topo IV could take over, we would expect an obligatory delay in cell division with a concomitant increase in cell size (assuming the rate of DNA replication is the limiting factor for cell division during rapid growth in rich medium). Upon microscopic examination, however, parE10 cells expressing γ alone that were grown at the permissive temperature were indistinguishable from cells expressing wild-type ParE. Additionally, the growth rates at the permissive temperature, based on OD, as well as viable CFU (data not shown) of parE10 cells expressing γ alone or wild-type ParE were similar. These data suggest that overexpression of γ does not significantly delay the average time between cell divisions and therefore probably does not slow DNA replication significantly.
Another interesting possibility is that γ, which has some characteristics of a chaperone, might be healing the damaged ParE protein at the nonpermissive temperature. This has been more difficult to test, because it requires the purification of the ParE10 protein, which, unlike the wild type, is insoluble when overexpressed (data not shown).
High-copy suppression of a temperature-sensitive allele is generally taken to indicate the existence of a complex between the two gene products, where the overexpressed protein stabilizes the temperature-sensitive protein at the nonpermissive temperature. However, we have been unable to detect an interaction between γ and ParE by gel filtration chromatography when the two proteins were mixed together at micromolar concentrations (data not shown). This does not eliminate the possibility of an interaction. The interaction between DnaG and DnaB at the replication fork, which can be detected functionally (51
) and by affinity chromatography (30
), cannot be detected by gel filtration.
The possibility of a physical interaction between γ and Topo IV could be eliminated if overexpression of dnaX
was found to suppress a parE
null allele, but to our knowledge no such allele exists, and our attempts to create one have been unsuccessful. However, neither pBR-dnaX
-γ can rescue the parC1215
) temperature-sensitive allele (data not shown). This suggests that γ expression is not compensating for a complete lack of Topo IV activity. It would be informative if dnaX
suppression of parE
was shown to be allele specific, but additional alleles are not known.
Given all this, our current working hypothesis is that γ and ParE interact. If this interaction occurs, it could take one of two forms. Topo IV could associate with the γ complex at the replication fork, or with free γ complex. γ complex can be isolated from the holoenzyme and may therefore exist in free form as well as forming part of the holoenzyme (33
). This excess γ complex may be essential for recycling β that is left behind on the nascent duplex DNA after the lagging-strand polymerase moves to a new primer during lagging-strand synthesis (58
). If there were an interaction between Topo IV and free γ complex, it would suggest a novel function for the latter.
In support of a Topo IV-γ interaction at the replication fork, interallelic suppressors of a temperature-sensitive parE
mutation in Salmonella typhimurium
have been mapped to dnaE
), encoding the α subunit of the holoenzyme (59
). If there is a Topo IV-replication fork interaction, why might this interaction take place?
Within the cell, Topo IV may not have free access to the replicating DNA. Double-stranded DNA binding proteins may limit the access of Topo IV to the DNA, and/or Topo IV may be sequestered in some manner from the replicating chromosome. Topo IV may be membrane associated, as suggested by the observation that under certain conditions of isolation ParC has been shown to be associated with the inner cell membrane (18
). As previously suggested, the membrane association of Topo IV may be via ParF, an inner membrane protein first identified in a screening for partition mutations (along with parE
) in Salmonella
). Additional support for the idea that Topo IV does not have free access to the replicating DNA is the observation that only gyrA
mutations show an immediate-stop DNA replication phenotype, as expected for the enzyme that supports this reaction in vivo (10
), even though Topo IV is as capable as DNA gyrase of supporting nascent chain elongation during theta-type DNA replication in vitro (12
) and there are roughly equivalent amounts of Topo IV and gyrase in the cell (about 400 tetramers [12
Association with the replication fork could serve as an entry point for Topo IV to the DNA. After association, Topo IV could ride along with the advancing replication fork or be dropped off behind the fork to relax the positive windings that arise between the newly replicated daughter duplexes. Alternatively, the interaction could take place between Topo IV and the replication fork as it is nearing completion of replication, forming a termination complex. A membrane-associated termination complex would be strategically positioned for signaling to partition or septation proteins that DNA replication and decatenation were complete.