Abstract:The ~sc~eric~ia cdi single-strands I3NA binding protein (SSB), essential for DNA ~e~~~at~~n, ~~~~b~~~~ lion and repair, can undergo a ~~~a~~~ induced irreversible ~onform~t~o~a~ change which does not ehminate its biological activity, but changes the number of nucieotides it covers (binding site size) when binding to a single-stranded nucleic acid httice. The binding site size of native srnd conformationally changed SSB was also found to be a function of the molecular mass of the polynucteotide, an observation … Show more
“…Even the highest amount of overproduced DinB in this report (about 80 μM; Fig. 5) would thus not effectively compete with SSB (1.7-3.4 μM; Bobst et al, 1985) in terms of ssDNA binding. Moreover, although DinB also interacts with SSB (Furukohri et al, 2012), we have not been able to detect replacement of SSB on SSB-coated ssDNA by DinB (AF, MTA and HM, unpublished results).…”
The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dosedependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.
“…Even the highest amount of overproduced DinB in this report (about 80 μM; Fig. 5) would thus not effectively compete with SSB (1.7-3.4 μM; Bobst et al, 1985) in terms of ssDNA binding. Moreover, although DinB also interacts with SSB (Furukohri et al, 2012), we have not been able to detect replacement of SSB on SSB-coated ssDNA by DinB (AF, MTA and HM, unpublished results).…”
The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dosedependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.
“…c Value reported is for the monomer. Other reported values range from 23,700 to 33,000 (43,44,149,188,191,220,223,326,350,422,423). See references 43 and 220 for a discussion.…”
“…Increasing the concentration of SSB resulted in an increased amount of SSB co-precipitating with Pol IV. Since the concentration of SSB tetramer in vivo is ∼1.7– 3.4 µM (29), we conclude that SSB is able to interact with Pol IV at concentrations that approximate to those found in normal cells. Figure 2.Pol IV and SSB interact directly in vitro .…”
DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.
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