Molecular mechanisms of holliday junction processing in Escherichia coli

doi:10.1016/0065-227X(95)99382-Y

Advances in Biophysics

Volume 31, 1995, Pages 49-65

https://doi.org/10.1016/0065-227X(95)99382-Y Get rights and content

Abstract

Recent genetic and biochemical studies revealed the mechanisms of late stage of homologous recombination in E. coli. A central intermediate of recombination called “Holliday structure”, in which two homologous duplex DNA molecules are linked by a single-stranded crossover, is formed by the functions of RecA and several other proteins. The products of the ruvA and ruvB genes, which constitute an SOS regulated operon, form a functional complex that promotes migration of Holliday junctions by catalyzing strand exchange reaction, thus enlarging the heteroduplex region. RuvA is a DNA-binding protein specific for these junctions, and RuvB is a motor molecule for branch migration providing energy by hydrolyzing ATP. The product of the ruvC gene, which is not regulated by the SOS system, resolves Holliday junctions by introducing nicks at or near the crossover junction in strands with the same polarity at the same sites. The recombination reaction is completed by sealing the nicks with DNA ligase, resulting in spliced or patched recombinants. The product of the recG gene provides an alternative route for resolving Holliday junctions. RecG has been proposed to promote branch migration in the opposite direction to that promoted by RecA protein. The atomic structure of RuvC protein revealed by crystallographic study, when combined with mutational analysis of RuvC, provides mechanistic insights into the interactions of RuvC with Holliday junction.

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Branched DNA with junction points has also been used as building blocks. A typical branched DNA forms a four-armed Holliday junction, an intermediate structure formed during the process of genetic recombination (Holliday, 1964; Shinagawa and Iwasaki, 1995). In addition, Y-shaped (Y-DNA) and X-shaped (X-DNA) DNAs have been designed and synthesized using three or four oligodeoxynucleotides (ODNs), respectively, with the halves of each ODN being partially complementary to a half of each of the other two ODNs (Kallenbach et al., 1983; Ma et al., 1986) (Fig. 1B).
Nucleic acids, DNA and RNA, not only allow transfer and replication of densely coded genetic information, but also act as danger signals triggering innate immune response. Recent progress in the design and construction of nano-sized structures using DNA has opened a new field of nanotechnology. The unique properties of nano-sized DNA constructs can be exploited to develop programmable materials for efficient delivery of bioactive compounds. In this review, recent advances in DNA nanotechnology and its applications as delivery systems are summarized. In particular, we focus on the delivery of DNA containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotide, or CpG motif, to immune cells expressing Toll-like receptor 9. Recent studies have shown that precisely designed DNA constructs, such as multi-branched DNA, polyhedral DNA, and DNA origami, can be used to enhance the biological activity of CpG DNA.
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The RuvABC proteins also function in a similar manner in the processing of Holliday junctions in bacteria. For instance, the RuvABC resolvasome complex promotes homologous recombination by migrating the Holliday junction to expand the heteroduplex region (reviewed in Shinagawa and Iwasaki, 1995; West, 1997). Of the three proteins, RuvB functions as a motor protein to move the junction point.
DNA and RNA frequently form various branched intermediates that are important for the transmission of genetic information. Helicases play pivotal roles in the processing of these transient intermediates during nucleic acid metabolism. The archaeal Hef helicase/ nuclease is a representative protein that processes flap- or fork-DNA structures, and, intriguingly, its C-terminal half belongs to the XPF/Mus81 nuclease family. Here, we report the crystal structure of the helicase domain of the Hef protein from Pyrococcus furiosus. The structure reveals a novel helical insertion between the two conserved helicase core domains. This positively charged extra region, structurally similar to the “thumb” domain of DNA polymerase, plays critical roles in fork recognition. The Hef helicase/nuclease exhibits sequence similarity to the Mph1 helicase from Saccharomyces cerevisiae; XPF/Rad1, involved in DNA repair; and a putative Hef homolog identified in mammals. Hence, our findings provide a structural basis for the functional mechanisms of this helicase/nuclease family.
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Holliday junctions occur as intermediates in homologous recombination and DNA repair. In bacteria, resolution of Holliday junctions is accomplished by the RuvABC system, consisting of a junction-specific helicase complex RuvAB, which promotes branch migration, and a junction-specific endonuclease RuvC, which nicks two strands. The crystal structure of a complex between the RuvA protein of M. leprae and a synthetic four-way junction has now been determined. Rather than binding on the open surface of a RuvA tetramer as previously suggested, the DNA is sandwiched between two RuvA tetramers, which form a closed octameric shell, stabilized by a conserved tetramer–tetramer interface. Interactions between the DNA backbone and helix-hairpin-helix motifs from both tetramers suggest a mechanism for strand separation promoted by RuvA.
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Background: Homologous recombination is crucial for genetic diversity and repairing damaged chromosomes. In Escherichia coli cells, the RuvA, RuvB and RuvC proteins participate in the processing of an important intermediate, the Holliday junction. The RuvA–RuvB protein complex facilitates branch migration of the junction, depending on ATP hydrolysis. The atomic structure of RuvA should enable critical questions to be addressed about its specific interactions with the Holliday junction and the RuvB protein.
Results: The crystal structure of RuvA shows the tetrameric molecules with a fourfold axis at the center. Each subunit consists of three distinct domains, some of which contain important secondary structure elements for DNA binding. Together with the detailed structural information, the biochemical assays of various mutant RuvA proteins and domains, isolated by partial proteolysis, allowed us to define the functional roles of these domains in Holliday junction binding and the RuvB interaction.
Conclusions: The RuvA molecule is formed by four identical subunits, each with three domains, I, II and III. The locations of the putative DNA-binding motifs define an interface between the DNA and the Holliday junction. Domain III is weakly attached to the core region, comprising domains I and II; the core domains can form a tetramer in the absence of domain III. Functional analyses of the mutant proteins and the partial digestion products, including Holliday junction binding and branch-migration assays, revealed that domain III and the preceding loop are crucial for RuvB binding and branch migration, although this region is not required for the junction–DNA binding.
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Molecular mechanisms of holliday junction processing in Escherichia coli

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