Reciprocal regulation between TOC1 and LHY/CCA1 within the Arabidopsis circadian clock.

The interactive regulation between clock genes is central for oscillator function. Here, we show interactions between the Arabidopsis clock genes LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), and TIMING OF CAB EXPRESSION 1 (TOC1). The MYB transcription factors LHY and CCA1 negatively regulate TOC1 expression. We show that both proteins bind to a region in the TOC1 promoter that is critical for its clock regulation. Conversely, TOC1 appears to participate in the positive regulation of LHY and CCA1 expression. Our results indicate that these interactions form a loop critical for clock function in Arabidopsis.

Biochemical characterization of the DNA helicase activity of the escherichia coli RecQ helicase.

We demonstrate that RecQ helicase from Escherichia coli is a catalytic helicase whose activity depends on the concentration of ATP, free magnesium ion, and single-stranded DNA-binding (SSB) protein. Helicase activity is cooperative in ATP concentration, with an apparent S(0.5) value for ATP of 200 microm and a Hill coefficient of 3.3 +/- 0.3. Therefore, RecQ helicase utilizes multiple, interacting ATP-binding sites to mediate double-stranded DNA (dsDNA) unwinding, implicating a multimer of at least three subunits as the active unwinding species. Unwinding activity is independent of dsDNA ends, indicating that RecQ helicase can unwind from both internal regions and ends of dsDNA. The K(M) for dsDNA is 0.5-0.9 microm base pairs; the k(cat) for DNA unwinding is 2.3-2.7 base pairs/s/monomer of RecQ helicase; and unexpectedly, helicase activity is optimal at a free magnesium ion concentration of 0.05 mm. Omitting Escherichia coli SSB protein lowers the rate and extent of dsDNA unwinding, suggesting that RecQ helicase associates with the single-stranded DNA (ssDNA) product. In agreement, the ssDNA-dependent ATPase activity is reduced in proportion to the SSB protein concentration; in its absence, ATPase activity saturates at six nucleotides/RecQ helicase monomer and yields a k(cat) of 24 s(-1). Thus, we conclude that SSB protein stimulates RecQ helicase-mediated unwinding by both trapping the separated ssDNA strands after unwinding and preventing the formation of non-productive enzyme-ssDNA complexes.

RecQ helicase and topoisomerase III comprise a novel DNA strand passage function: a conserved mechanism for control of DNA recombination.

E. coli RecQ protein is a multifunctional helicase with homologs that include the S. cerevisiae Sgs1 helicase and the H. sapiens Wrn and Blm helicases. Here we show that RecQ helicase unwinds a covalently closed double-stranded DNA (dsDNA) substrate and that this activity specifically stimulates E. coli topoisomerase III (Topo III) to fully catenate dsDNA molecules. We propose that these proteins functionally interact and that their shared activity is responsible for control of DNA recombination. RecQ helicase has a comparable effect on the Topo III homolog of S. cerevisiae, consistent with other RecQ and Topo III homologs acting together in a similar capacity. These findings highlight a novel, conserved activity that offers insight into the function of the other RecQ-like helicases.

RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination.

RecQ helicase is important to homologous recombination and DNA repair in Escherichia coli. We demonstrate that RecQ helicase, in conjunction with RecA and SSB proteins, can initiate recombination events in vitro. In addition, RecQ protein is capable of unwinding a wide variety of DNA substrates, including joint molecules formed by RecA protein. These data are consistent with RecQ helicase assuming two roles in the cell; it can be (1) an initiator of homologous recombination, or (2) a disrupter of joint molecules formed by aberrant recombination. These findings also shed light on the function of the eukaryotic homologs of RecQ helicase, the Sgs1, Blm, and Wrn helicases.

Interaction of Escherichia coli RecA protein with LexA repressor. II. Inhibition of DNA strand exchange by the uncleavable LexA S119A repressor argues that recombination and SOS induction are competitive processes.

The Escherichia coli RecA protein is involved in SOS induction, DNA repair, and homologous recombination. In vitro, RecA protein serves as a co-protease to cleave LexA repressor, the repressor of the SOS regulon; in addition, RecA protein promotes homologous pairing and DNA strand exchange, steps important to homologous recombination and DNA repair. To determine if these two functions of RecA protein are competing or parallel, the effect of uncleavable LexA S119A repressor on RecA protein-dependent activities was examined. LexA S119A repressor inhibits both the single-stranded DNA (ssDNA)-dependent ATP hydrolysis and DNA strand exchange activities of RecA protein. As for wild-type LexA repressor (Rehrauer, W. M., Lavery, P. E., Palmer, E. L., Singh, R. N., and Kowalczykowski, S. C. (1996) J. Biol. Chem. 271, 23865-23873), inhibition of ATP hydrolysis is dependent upon the presence of E. coli single-stranded DNA binding (SSB) protein, arguing that LexA repressor affects the competition between RecA protein and SSB protein for ssDNA binding sites. In contrast, inhibition of DNA strand exchange activity is SSB protein-independent, suggesting that LexA S119A repressor blocks a site required for DNA strand exchange. These results imply that there is a common site on the RecA protein filament for secondary DNA and LexA repressor binding and raise the possibility that the recombination and co-protease activities of the RecA protein filament are competitive.