DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

Abstract

DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flashcooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. D NA is frequently considered a passive component in interactions with proteins involved in DNA metabolism. Despite this view, many proteins use DNA structural features to mediate catalysis and identify damaged DNA through the effects of damage on DNA rigidity and conformation (1-6). The view of DNA as a passive element is therefore at least in part due to a paucity of robust tools to examine dynamic DNA conformational states during multistep reactions. Gold-labeled DNA enables measurement over length scales sufficient to accommodate several proteins to identify cooperative effects on DNA. Because X-rays scatter predominantly from electrons, using heavy atom labels (7-11) provides high contrast relative to organic molecules. By using labels of moderate size (∼5 nm), the scattering from gold nanocrystals dominates all other scattering signals by three orders of magnitude, thereby reducing analysis complexity while minimizing nanocrystal influence on biological macromolecules. Importantly, small angle X-ray scattering (SAXS) provides global information on conformations adopted by a population of macromolecules in almost any solution condition (12)(13)(14).Mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication (15,16). Despite the i...

MutL traps MutS at a DNA mismatch

“…This latter finding provides an explanation for the observation that yMutLα can interact with an ATPasesite mutant of yMutSα that does not form a sliding clamp (30). Interestingly, a recent study monitoring DNA bending with small angle X-ray scattering in solution (31), suggests that, for E. coli proteins, MutL interacts with MutS after an ATP-dependent conformational change from a bent DNA state to an unbent DNA state. If E. coli and Taq MMR follow the same pathway (discussed later in Conclusions), then extrapolating this result suggests that our FRET 0.45 state (between protein and DNA) involves unbent DNA (Fig.…”

From a structural average to the conformational ensemble of a DNA bulge

“…X-ray interferometry readily provides information about translational displacements, which are difficult to assess with RDC measurements (47), as well as angular movements, and it is more straightforward to extend to larger structures (22,24) and other classes of macromolecules. However, X-ray interferometry is limited in detecting rare conformers (17,18,23) so that techniques that can trap or assess rare excursions, such as NMR relaxation dispersion (48), paramagnetic relaxation enhancement (49), hydrogen/deuterium exchange (50), and cyclization (51), are powerful complements to X-ray interferometry.…”

“…X-ray interferometry can be used to determine site-tosite distance distributions instantaneously because it relies on atomic scattering (17,(19)(20)(21)(22)(23)(24). Standard small-angle X-ray scattering (SAXS) measures the sum of the scattering and scattering interference from all atoms in a macromolecule (25).…”

New insights into the mechanism of DNA mismatch repair

“…On the other hand, the sliding clamp model proposes that the mispair is recognized by an ADP-bound MutS, resulting in a rapid ADP-ATP exchange and a MutS conformational change that releases MutS from the mismatch allowing MutS to diffuse along the DNA backbone as a sliding clamp, in an ATP-hydrolysis-independent manner (Acharya et al 2003; Gradia et al 1999; Hura et al 2013; Lee et al 2014; Mendillo et al 2005). DXMS studies have revealed some of these ATP binding induced conformational changes (Mendillo et al 2010).…”