DNA Repeat Rearrangements Mediated by DnaK-Dependent Replication Fork Repair

Abstract

We propose that rearrangements between short tandem repeated sequences occur by errors made during a replication fork repair pathway involving a replication template switch. We provide evidence here that the DnaK chaperone of E. coli controls this template switch repair process. Mutants in dnaK are sensitive to replication fork damage and exhibit high expression of the SOS response, indicative of repair deficiency. Deletion and expansion of tandem repeats that occur by replication misalignment ("slippage") are also DnaK dependent. Because mutations in dnaX encoding the gamma and tau subunits of DNA polymerase III mimic dnaK phenotypes and are genetically epistatic, we propose that the DnaKJ chaperone remodels the replisome to facilitate repair. The fork remains largely intact because PriA or PriC restart proteins are not required. We also suggest that the poorly defined RAD6-RAD18-RAD5 mechanism of postreplication repair in eukaryotes occurs by an analogous mechanism to the DnaK template-switch pathway in prokaryotes.

A DNA damage response in Escherichia coli involving the alternative sigma factor, RpoS

“…4A). Although recA is more highly expressed than dinB, both promoters are induced by all 4 DNA damaging treatments, as we have shown previously for AZT (19). These results suggest that expression of IraD is induced transcriptionally not only after oxidative stress (13) but after other forms of DNA damage.…”

“…4B), suggesting that the DNA damage responsiveness of the iraD promoter is not mediated via the SOS response. In control experiments, induction of the recA promoter by these agents was largely abolished by recA or lexA mutations (data not shown) (19). The SOS independence of IraD induction was not unexpected, given its failure to respond to MitoC and the lack of apparent LexA binding consensus sites in the upstream region of iraD.…”

“…Luciferase reporter assays were performed as described (19). After transformation into MG1655, lexA3, or recA::cat strains, the promoter assays were performed with exponentially growing cultures (OD600 0.2-0.4) exposed to 20 g/mL phleomycin, 25 mg/L hydrogen peroxide, 1 g/mL AZT, or 1.5 g/mL mitomycin C for 40 min.…”

Control of the function of the transcription and repair factor TFIIH by the action of the cochaperone Ydj1

“…Evidence for the conformational changes of protein complexes at their site of action has been provided for nuclear proteins (9,10), and our study proposes TFIIH as a nuclear substrate of chaperones. Physical interactions between Ydj1 and TFIIH components had been previously reported for Rad3 (16) and Tfb2 (17).…”

“…Hsp90 also promotes replication through UV-damaged templates in human cells (9). Another link between chaperones and replication was previously reported in Escherichia coli for the Hsp70 cochaperone homologue when template switching was needed to accomplish repair (10). Interestingly, chaperones have also been related to NER, as genetic and physical interactions were described between Hsp90-Sti1 and the other TFIIH helicase, Rad25 (11), and synthetic lethal interactions were found between the TFIIH kinase, Kin28, and the cochaperones Sti1 and Cdc37 (12).…”