DNA is susceptible to a variety of mutations and chemical modifications. Ionization radiation causes double strand breaks in DNA, and chemical modifications potentially lead to double strand breaks during replication. A major pathway to repair double-strand breaks is homologous recombination. Robin Holliday first proposed a four-way DNA junction in 1964 as an intermediate in recombination between two homologous DNA duplexes to account for gene conversion. The Holliday junction (HJ) has since been identified as a central molecular species in DNA recombination and repair. Phage T4 endonuclease VII (Endo VII) was the first enzyme shown to resolve HJs into duplex DNAs by introducing symmetrical nicks in either pair of equivalent strands. Several HJ resolvases have since been characterized, but an atomic structure of a resolvase complex with an HJ DNA had remained elusive.[unreadable] [unreadable] My group picked up the project of structural studies of T4 endo VII when Dr. Christian Biertmpfel joined us at NIH. Dr. Biertmpfel obtained his doctoral degree at EMBL (Heidelberg, Germany) and there he grew many crystals of Endo VII complexed with HJs. But most of them didnt diffract X-rays well. Only one crystal yielded diffraction data to near 3 , but its structure could not be solved. At NIH, after re-designing DNA junctions as substrate for Endo VII, we were able to grow new crystals of Endo VII-DNA complexes and solve the new crystal form by molecular replacement. Using the new crystals grown at NIH, Dr. Biertmpfel eventually solved the old crystal of HJ-Endo VII complexes grown in Heidelberg. [unreadable] [unreadable] The crystal structure is of an inactive Endo VII (N62D) complexed with an immobile four-way junction with alternating 10 and 14 bp arm lengths. The Holliday junction is a hybrid of the conventional square-planar and stacked-X conformation. Endo VII binds the HJ with its predominantly positively charged face and protrudes into the junction point from the minor groove side, opening it to a 14 X 31 parallelogram. This interaction interrupts the coaxial stacking, yet every base pair surrounding the junction remains intact. Additional interactions between the protein and HJ involve the phosphate backbones only. Each scissile phosphate two base pairs from the crossover interacts with a Mg2+ ion in the active site. The similar overall shape and surface charge potential of the HJ resolvases Endo VII, RuvC, Ydc2, Hjc and RecU, despite their very different fold and active site sequences, suggest a conserved binding mode for four-way junctions. The results have been reported in a Letter to Nature, currently in press.[unreadable] [unreadable] Ini this fiscal year, we set out to improve the resolution of the Endo VII-junction complexes, to find out how the metal ion-dependent catalysis occurs and to identify new junction resolvases. So far our experimental efforts have met little success. This may be the last year that we work on the Holliday junction resolvase project.
NIH ExPorter
NIH ExPorter