Crystal structure of RuvC resolvase in complex with Holliday junction substrate

• Published in: Nucleic acids research Vol. 41; no. 21; pp. 9945 - 9955
• Main Authors: Górecka, Karolina M, Komorowska, Weronika, Nowotny, Marcin
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.11.2013
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Crystallography, X-Ray; DNA, Cruciform - chemistry; DNA, Cruciform - metabolism; Holliday Junction Resolvases - chemistry; Holliday Junction Resolvases - metabolism; Models, Molecular; Molecular Sequence Data; Structural Biology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkt769

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.