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Proteins Specifically Interact with Holliday Junctions and Promote Branch Migration

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Proteins Specifically Interact with Holliday Junctions and Promote Branch Migration Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Escherichia coli RuvA and RuvB proteins specifically interact with Holliday junctions and promote branch migration Hiroshi Iwasaki, Masahiko Takahagi, Atsuo Nakata, and Hideo Shinagawa 1 Department of Experimental Chemotherapy, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan The Escherichia coli ruvA and ruvB genes are involved in DNA repair and in the late step of homologous genetic recombination. We have demonstrated previously that the RuvA-RuvB protein complex in the presence of ATP promotes reabsorption of cruciform structures extruded from a supercoiled plasmid with an inverted repeat sequence. Because the cruciform structure is topologically analogous to the Holliday structure, we have proposed that the role of the RuvA and RuvB proteins in recombination is to promote a strand exchange reaction at the Holliday junction. Here, we studied the specific interaction of the RuvA-RuvB complex with the Holliday structure using synthetic analogs prepared by annealing four oligonucleotides. The affinities of the RuvA protein for synthetic Holliday junctions are much higher (>20-fold) than for duplex DNA, and the affinities of the RuvA protein for the junctions are further enhanced (>4-fold) by the interaction with the RuvB protein. The RuvA-RuvB protein complex in the presence of ATP promotes dissociation of the synthetic Holliday junction with homology in the central core into two halves by catalyzing branch migration to the DNA ends, but it does not affect the structure of the synthetic Holliday junction without the homology. The separation of the synthetic Holliday junction is a result of the activity of the RuvA-RuvB complex that promotes strand exchange and DNA unwinding. Furthermore, RuvA and RuvB promote the strand exchange reaction at the Holliday junctions made by RecA. These results provide further evidence that the RuvA-RuvB complex recognizes the Holliday junction and promotes branch migration in homologous recombination. [Key Words: Branch migration; helicase; Holliday junction; recombination; RecA protein; RuvA-RuvB protein complex] Received June 26, 1992; revised version accepted August 27, 1992. Escherichia coli cells with a mutation in any of the three and ruvB genes constitute a LexA-regulated operon ruv genes, ruvA, ruvB, and ruvC, are sensitive to various [Shurvinton and Lloyd 1982; Benson et al. 1988; Shina- DNA-damaging agents such as UV irradiation, X-rays, gawa et al. 1988), whereas the ruvC gene located near the and chemical mutagens (Otsuji et al. 1974; Lloyd et al. ruvA-ruvB operon is not regulated by the SOS system 1984; Iwasaki et al. 1989a; Sargentini and Smith 1989; (Sharpies et al. 1990; Sharpies and Lloyd 1991; Takahagi Sharples et al. 1990). Although the ruv single mutants et al. 1991). Like the recA gene, the ruvA and ruvB genes are nearly as proficient as the wild-type strain in genetic apparently play multiple roles in recombination, repair, recombination, the ruv derivatives of recB C sbcA, recB C and mutagenesis. sbcBC, and recG are severely defective in recombination The RuvC protein has been purified recently and and highly sensitive to DNA-damaging agents (Lloyd shown to be an endonuclease that resolves the Holliday 1991; Lloyd et al. 1984, 1987). These findings suggest structure (Dunderdale et al. 1991; Iwasaki et al. 1991), that the products of ruvA, ruvB, and ruvC are involved in which is a proposed intermediate of homologous recom- recombination repair of damaged DNA. In addition, the bination (Holliday 19641. The RuvA and RuvB proteins ruvA and ruvB mutants have some defects in X-ray- and have been overproduced and purified to near homogene- UV-induced mutagenesis (Sargentini and Smith 1989; H. ity, and their properties have been studied (Iwasaki et al. Iwasaki, T. Kato, and H. Shinagawa, unpubl.). The ruvA 1989b; Shiba et al. 1991). The RuvB protein possesses weak ATPase activity, which is greatly enhanced by the RuvA protein bound to DNA. In the presence of ATP, 1Corresponding author. the RuvA-RuvB protein complex promotes reabsorption 2214 GENES & DEVELOPMENT 6:2214-2220 © 1992 by Cold Spring Harbor Laboratory ISSN 0890-9369/92 $3.00 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press of a cruciform structure that is formed by extrusion of an 1-5). The affinity of the RuvA protein for the synthetic inverted repeat sequence in supercoiled DNA. Because Holliday junction was 20-fold or more than that for the the cruciform structure is structurally analogous to the duplex DNA, as estimated by the amount of RuvA Holliday structure in which two homologous duplex needed to give equivalent binding. molecules are linked by a single-stranded crossover, and the reabsorption of the cruciform involves exchanges of single-stranded partners between the homologous pairs RuvB protein enhances the affinity of RuvA protein for the Holliday junction of duplex DNA, we proposed previously that the role of the RuvA and RuvB proteins in homologous recombina- As the DNA-bound RuvA protein enhances the ATPase tion is to interact specifically with the Holliday junction activity of the RuvB protein (Shiba et al. 1991), we and promote branch migration (Iwasaki et al. 1991; Shiba wanted to determine whether RuvB enhances DNA- et al. 1991; Shinagawa et al. 1991). binding activity of RuvA. As shown in Figure 2A, RuvA In this work we studied specific interactions of the in the presence of excess RuvB exhibited approximately RuvA protein and the RuvA-RuvB protein complex with fourfold higher affinity for the junction DNA than it did synthetic Holliday junctions and with the Holliday in the absence of RuvB. Because a RuvA tetramer forms structures made by RecA and obtained further evidence a stable complex with a RuvB dimer in solution (Shina- that supports the above hypothesis. gawa et al. 1991), and the relative electrophoretic mobil- ities of the protein-DNA complexes formed in the pres- ence and in the absence of the RuvB protein were the Results same, the RuvB protein appears to be dissociated from RuvA protein binds preferentially the RuvA-DNA complex during electrophoresis under to the synthetic Holliday junction the present experimental conditions. RuvB also appears to enhance the affinity of RuvA for the duplex DNA (Fig. Because the RuvA-RuvB protein complex promotes, 2A, lanes 6-10). with accompanying ATP hydrolysis, reabsorption of the cruciform extruded from supercoiled DNA with an in- verted repeat, and the RuvA protein itself is a DNA- Effect of the homology core in the junction on the binding protein (Shiba et al. 1991), we wanted to show interaction with the RuvA-RuvB protein complex that RuvA actually binds to the Holliday junction. We The effect of the homology core in the junction on the prepared synthetic four-way junctions by annealing four interaction with the RuvA-RuvB complex was studied nucleotide oligomers as analogs of the Holliday junction by comparing relative affinities of the synthetic Holliday {Fig. 1) and examined by gel-retardation assay whether junctions with and without a homology core for the pro- the RuvA protein binds preferentially to the Holliday tein complex. The RuvA-RuvB complex showed similar junction with a central homology core of 12 bp (Fig. 2A). affinity for the junction DNA with or without the ho- RuvA formed complexes with the Holliday junction (Fig. mology core in the repeated experiments. It bound to 2A, lanes 11-15) in the concentration range that allowed both of the junction DNAs with much higher affinity no complex formation with the linear duplex DNA (lane (-20-fold) than to the duplex DNAs with or without the inverted repeat sequence (Fig. 2B). Therefore, the RuvA- RuvB protein complex interacts preferentially with Hol- liday junctions, and the strength of this interaction ap- oligo 12 34 15 67 89 1011 pears to be largely dependent on the topology of DNA. A B A B A A The RuvA-RuvB protein complex dissociates the Holliday junction by promoting branch migration We examined the effect of nucleotides on the interaction between the RuvA-RuvB protein complex and the syn- thetic Holliday junction by gel retardation assay. No ef- D C D D fect of nucleotides ATP, ADP, or ATP(~/S) on the binding affinity of the RuvA-RuvB protein complex for the junc- I II I IV tion was observed (data not shown). However, a new DNA band, which migrated faster than the junction Figure 1. Schematic drawing of synthetic Holliday junctions DNA, was observed by incubation with ATP but not and duplex DNAs used in this study. One of the synthetic Hol- with ADP or ATP(~/S). We then analyzed by gel electro- liday junctions (I) contains a homology core of 12 bp (solid bars) flanked by heterologous sequences A, B, C, and D, which are phoresis the DNA products after the reaction products shared by the Holliday junction that does not contain homolo- were treated with EDTA and SDS to dissociate protein gous sequences (II). The duplex DNA with inverted repeat se- from DNA (Fig. 3A). The position of the DNA band (lane quences (III) contains a 12-bp inverted repeat (solid bars) flanked 7) between the junction and the duplex DNA bands cor- by A and D sequences, which are shared with the duplex DNA responded to that of a half-cruciform (marker 3 in lane 3). without inverted repeat sequences (IV). Therefore, we concluded that incubation of the junction GENES & DEVELOPMENT 2215 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press A duplex DNA synthetic Holliday no RuvB + RuvB no RuvB + RuvB o o o -bound Figure 2. Gel-retardation assay of RuvA binding to Holliday junctions.
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