Book of Abstracts: Albany 2005
Damaged DNA and Repair Machines: Damage Recognition, Conformational Changes and the Structural Chemistry for DNA Repair Coordination
To help define structures for dynamically assembled molecular complexes acting in genome integrity, we have built the Structurally Integrated Biology for Life Sciences (SIBYLS) beamline at the Advanced Light Source. SIBYLS provides technologies to characterize the structures, conformations, architectural arrangements, and assemblies of molecular machines acting in DNA direct damage reversal and DNA repair.
DNA genetic integrity for base-excision repair and replication depends upon the structure-specific repair and replication nuclease Flap EndoNuclease (FEN-1) and the trimeric processivity factor PCNA. To clarify the molecular basis of FEN-1 specificity and PCNA activation, we solved structures of FEN-1:DNA and PCNA:FEN-1-peptide complexes, along with fluorescence resonance energy transfer (FRET) and mutational results. FEN-1 binds the unpaired 3' DNA end (3' flap), opens and kinks the DNA, and promotes conformational closing of a flexible helical clamp to facilitate 5' cleavage specificity. Ordering of unstructured C-terminal regions in FEN-1 and PCNA creates an intermolecular β-sheet interface that directly links adjacent PCNA and DNA binding regions of FEN-1 and suggests how PCNA stimulates FEN-1 activity. Our proposed interface exchange hypothesis for coordinated transfer of DNA intermediates during PCNA-mediated processes helps explain how FEN-1 acts to avoid repeat expansions and micro satellite instability.
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. To clarify RAD51 interactions and assembly, we have determined the crystal structure and assembly of full-length RAD51. A polymerization motif tethers individual subunits into assemblies. A RAD51 filament assembly from 3D EM reconstructions and crystallographic interfaces suggests the basis for DNA interactions and supports a molecular mechanism for the ordered interactions of HRR partners by exchanges of RAD51 polymer interface elements.
John A. Tainer
Lawrence Berkeley National Lab