To clarify functions of the Mre11/Rad50 (MR) complex in DNA double-strand break repair, we report Pyrococcus furiosus Mre11 crystal structures, revealing a protein phosphatase-like, dimanganese binding domain capped by a unique domain controlling active site access. These structures unify Mre11's multiple nuclease activities in a single endo/exonuclease mechanism and reveal eukaryotic macromolecular interaction sites by mapping human and yeast Mre11 mutations. Furthermore, the structure of the P. furiosus Rad50 ABC-ATPase with its adjacent coiled-coil defines a compact Mre11/Rad50-ATPase complex and suggests that Rad50-ATP-driven conformational switching directly controls the Mre11 exonuclease. Electron microscopy, small angle X-ray scattering, and ultracentrifugation data of human and P. furiosus MR reveal a dual functional complex consisting of a (Mre11)2/(Rad50)2 heterotetrameric DNA processing head and a double coiled-coil linker.

O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine. The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.

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To understand how proteins translate the energy of sunlight into defined conformational changes, we have measured the photocycle reactions of photoactive yellow protein (PYP) using time-resolved step scan Fourier transform infrared (FTIR) spectroscopy. Global fit analysis yielded the same apparent time constants for the reactions of the chromophore, the protonation changes of protein side chains and the protein backbone motions, indicating that the light cycle reactions are synchronized. Changes in absorbance indicate that there are at least four intermediates (I1, I1', I2, I2'). In the intermediate I1, the dark-state hydrogen bond from Glu 46 to the aromatic ring of the p-hydroxycinnamoyl chromophore is preserved, implying that the chromophore undergoes trans to cis isomerization by flipping, not the aromatic ring, but the thioester linkage with the protein. This excludes an I1 structural model proposed on the basis of time resolved Laue crystallography, but does agree with the cryotrapped structure of an I1 precursor.