Abstract
The key intermediate in genetic recombination is the Holliday junction (HJ), a four-way DNA structure. At the end of recombination, HJs are cleaved by specific nucleases called resolvases. In Gram-negative bacteria, this cleavage is performed by RuvC, a dimeric endonuclease that belongs to the retroviral integrase superfamily. Here, we report the first crystal structure of RuvC in complex with a synthetic HJ solved at 3.75 Å resolution. The junction in the complex is in an unfolded 2-fold symmetrical conformation, in which the four arms point toward the vertices of a tetrahedron. The two scissile phosphates are located one nucleotide from the strand exchange point, and RuvC approaches them from the minor groove side. The key protein–DNA contacts observed in the structure were verified using a thiol-based site-specific cross-linking approach. Compared with known complex structures of the phage resolvases endonuclease I and endonuclease VII, the RuvC structure exhibits striking differences in the mode of substrate binding and location of the cleavage site.
Highlights
Genetic recombination is used to rearrange genes between homologous sequences and generate genetic diversity and promote evolution
After Holliday junction (HJ), arms corresponding to 3 and 4 in the complex structure are exchanged with arms corresponding to 1 and 2 (Figure 1)—an probable binding mode, the phosphate group of residue 28 is in the vicinity of M108
On the addition of the Tt-RuvC T11C variant to asymmetrically modified HJ-29bb-h or the P40C variant to HJ-28bb-h, the pattern of cross-linked products corresponded to the DNA alone but with lower mobility that resulted from attachment of the protein
Summary
Genetic recombination is used to rearrange genes between homologous sequences and generate genetic diversity and promote evolution. We prepared a bias-free model by superimposing the unliganded form of Tt-RuvC on the protein subunits in the final complex structure and fragments of ideal B-form DNA on the HJ arms with small rigid body adjustments of the positions of individual nucleotides.
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