Abstract
To elucidate the molecular mechanism of the resolution of Holliday junctions by Escherichia coli RuvC protein, we studied biochemical properties of the protein using various synthetic DNA junctions as model substrates. RuvC cleaves not only a four-way junction but also three-way junctions efficiently. The central core of homology in the junction is essential for the substrates to be cleavable by RuvC. Although the divalent cations are essential for the endonuclease activity, RuvC efficiently forms specific complexes with four-way junctions in the absence of the cations, irrespective of the presence of homologous core sequences. By using T7 endonuclease I as a probe, we studied the topology of the substrate junctions used in our study. The results suggest that RuvC cleaves the three-way junctions with homology core when they become four-way conformers. From the present studies, we propose that RuvC initially binds mostly nonproductively to four-way junctions, which does not require divalent metals, and subsequently cleaves the junctions by a mechanism dependent on a divalent cation and a particular topological conformer that is induced by the sequences at the mobile junctions.
Highlights
To elucidate the molecular mechanism of the resolu- 1992)
RuvC protein has beenpurified andshown to be an tion of Hollidajyunctions by Escherichia coli RuvC pro- endonuclease that cleavesHolliday junctions by tein, we studied biochemical properties of the protein introducing nicks into DNA strands with the same polarity at using various synthetic DNA junctions as model sub- or near the junction
These oligonucle- Holliday Junction by RuvC-For quantitativeassay of the otides were purified by reverse phase column chromatography with RuvC resolvase activity, a four-way junction with homologous high performance liquid chromatography.All oligonucleotides werere- core sequences of 12 base pairbsetween pairs of the arms (4Jh) suspended in TE buffer (10 mM Tris-HC1, pH 8.0, 1mM EDTA)
Summary
Co2+at a higher concentration (25 mM) was moderately effective for the activity These resultsalso implythat divalentcations such as Mg2' are cleavage of synthetic junctions (which contained RuvC (11nM) or T7 required for the cleavage reaction at the step other than the endonuclease I (20 nM) and 32P-labeledsubstrate DNA (0.3 p ~ in) the buffer containing 20 mM HEPES-KOH, pH 7.5, 8 m~ magnesium acetate, 10mM potassium glutamate, and10%glycerol)was incubated for 5 min a t 37 "C.Reactionswere stoppedby the addition of 4pl of loading buffer(20 mM HEPES-KOH,pH 7.5, 10 mM EDTA, 0.1% SDS, 20% glycerol, and 0.05%bromphenol blue), and the reaction products were formation of the stacked X-structuresince Mg2' and Ca2+were effective in forming this structure (Duckett aelt., 1990) and Ca2+was much less effective for the endonuclease activity of RuvC than M e. RuvC Cleavage Sites in the Four-way Junction-We determined the RuvC cutting sites indTh by analyzing thecleaved was detected with 35, which had no homology around the junc- products on sequencing gels and compared the cleavage pattern tion (lanes 7 and 8).3Jh was cleaved as fast as4Jh (Fig. 1C). C, mapping of the T7 endonuclease I cleavage sites in and 3Jh
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
