Abstract
Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.
Highlights
To cite this version: Marie Agnes Petit, Wendy Bedale, Jerzy Osipiuk, Chi Lu, Malini Rajagopalan, et al
&plication-blocking lesions generateasignal in with RecA and DNA polymerase 111holoenzyme, promotes in Escherichia coli that leads to the induction ofthe mul- vitro replication across the sitoef the DNA lesion (Rajagopalan tigene SOS response.Among the SOS-inducedgenes are et al, 1992)
Results obtained with anti-UmuC antibodies are presented with GroEL and GroES only without prior treatmentwith the was visible when UmuC had been treated with DnaK, DnaJ, Immunoblotting experiment withGroEL antibodies allowed and GrpE (Fig. L 4, lane CJKE)
Summary
UmuC is anE. coli protein that wasfound to requirethe help UmuC in buffer B (20mM Hepes pH 8,100 mM potassium acetate, 8 mM of a chaperone-like protein (the ribosomalprotein S9) tobe maintained in a soluble and active form (Woodgateet al., 1989). At 30 “Cin a10-plreaction containing 20 mM Tris-C1, pH 7.5,8 mM MgCl,, this point, UmuC was applied to and eluted from a second phosphocel- 5 mM dithiothreitol, 0.1 mM EDTA, 25nm sodium glutamate, 1mM ATP, lulose columnwith a buffer containing 7 M urea and 500 mM KC1 This 4% (v/v)glycerol, 40 pg/ml bovine serum albumin, 5% (w/v) PEG 6000, fraction was diluted 2.5-fold into K Buffer (10mM KPO,, pH 6.8, 5 mM 2.5 nM primed DNA substrate, 3100 m single-stranded DNA binding octyl P-o-thioglucopyranoside, mM glutathione, 4% glycerol)and dia- protein, 0.1 m p protein, 2500 nM RecA803, and 1300 nM UmuD’. Other proteins used in theassays were purified as described: UmuD’ (Woodgate et al, 1989),DNA polymerase I11 holoenzyme (Maki et al, 1988),DnaJ (Zylicz etal., 19851,DnaK (Zyliczand Georgopoulos, 1984), GrpE (Zylicz et al, 1987), GroEL and GroES (Chandrasekhar et al, 1986).
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