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Envisioning new targets and new approaches for molecular-based cancer therapeutics

We aim to understand how genome maintenance and stress responses are coordinated by dynamically changing, multiprotein complexes. Reversible complexes involve composite interfaces from modular preformed and unstructured regions that provide strong, specific contacts from the combination of relatively weak, modular interactions. These individual modular interaction sites, which allow protein exchanges and pathway progression, also provide possible targets for new therapeutic strategies. DNA genetic integrity and cancer avoidance depends upon the structure-specific repair and replication nuclease flap endonuclease (FEN-1) and upon the trimeric processivity factor PCNA. FEN-1 and PCNA complex structures and mutational results provide a coherent model for DNA substrate recognition and PCNA activation of FEN-1. Together, these structural and mutational results support an interface exchange hypothesis for coordinated transfer of DNA intermediates during PCNA-mediated processes. We have furthermore defined analogous interface exchange as an important coordinating factor for homologous recombination repair (HRR) of DNA double-strand breaks. The Mre11/Rad50 (MR) complex that first recognizes DNA double-strand breaks and Rad51 complexes that promote recombination are essential for DNA break repair and recombination processes. To help understand the molecular mechanism of the MR complex in DSB repair, we determined crystal structures of Mre11 and Rad50 catalytic domains (Mre11cd and pfRad50cd), their interface, DNA interactions, and a unique Zn-hook linking the 600 Å coiled-coil domain of Rad50. The MR complex must handoff the DNA ends to Rad51, which catalyzes homologous pairing and exchange between dsDNA and ssDNA via orchestrated interactions with BRCA2, Rad52, and other HRR proteins. We determined an atomic structure of a polymeric full-length RAD51 homolog to reveal atomic details of quaternary assembly, we experimentally test a proposed BRC repeat-induced RAD51 disassembly mechanism and we address the molecular mechanism for the orchestrated interactions of RAD51 in HRR. The Rad51 structure reveals a polymerization motif involving an interdomain linker key for quaternary assembly. Structural and mutational results suggest how differences in RAD51 ring and helical nucleoprotein filament assemblies may regulate ATPase activity. A RAD51 filament assembly based on 3D EM reconstructions and crystallographic interfaces suggests a novel role for RAD51 N-terminal domains in binding dsDNA within a large outer groove. By taking advantage of the simpler organization of archaeal recombination systems, our structural and mutational results in conjunction with HsRAD51-AD:BRC4 results establish at the molecular level how BRC repeats disrupt RAD51 assembly and direct RAD51 to form foci in cells in response to DNA damage. Our results help support a molecular mechanism for the ordered interactions of HRR partners BRCA2, RAD52, RAD54 and RAD55 by protein-mediated and DNA-mediated exchanges of RAD51 polymer interface elements. To achieve accurate structural information on these difficult but biologically relevant molecular complexes, we have designed and developed the Structurally Integrated Biology for Life Sciences beamline at the Advanced Light Source. This provides a unique resource for X-ray diffraction characterizations of both static and time-resolved solution states of macromolecular machines.

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Tainer, J. Envisioning new targets and new approaches for molecular-based cancer therapeutics. Breast Cancer Res 7 (Suppl 2), S.44 (2005).

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