No abstract

DNA polymerase β (pol β) fills single nucleotide gaps in DNA during base excision repair and non-homologous end-joining. Pol β must select the correct nucleotide from among a pool of four nucleotides with similar structures and properties in order to maintain genomic stability during DNA repair. Here, we use a combination of X-ray crystallography, fluorescence resonance energy transfer and nuclear magnetic resonance to show that pol β‘s ability to access the appropriate conformations both before and upon binding to nucleotide substrates is integral to its fidelity. Importantly, we also demonstrate that the inability of the I260Q mutator variant of pol β to properly navigate this conformational landscape results in error-prone DNA synthesis. Our work reveals that precatalytic conformational rearrangements themselves are an important underlying mechanism of substrate selection by DNA pol β.

Analysis of complex mixtures of proteins by hydrogen exchange (HX) mass spectrometry (MS) is limited by one's ability to resolve the protein(s) of interest from the proteins that are not of interest. One strategy to overcome this problem is to tag the target protein(s) to allow for rapid removal from the mixture for subsequent analysis. Here we illustrate a new solution involving fluorous conjugation of a retrievable probe. The appended fluorous tag allows for facile immobilization on a fluorous surface. When a target protein is passed over the immobilized probe molecule it can be efficiently captured and then exposed to a flowing stream of deuterated buffer for hydrogen exchange. The utility of this method is illustrated for a model system of the Elongin BC protein complex bound to a peptide from HIV Vif. Efficient capture is demonstrated and deuteration when immobilized was identical to deuteration in conventional solution-phase hydrogen exchange MS. Protein captured from a crude bacterial cell lysate could also be deuterated without need for separate purification steps before HX MS. The advantages and disadvantages of the method are discussed in light of miniaturization and automation.

The single-stranded DNA (ssDNA) binding protein RPA binds to and protects ssDNA while simultaneously recruiting numerous replication and repair proteins essential for genome integrity. In this issue of Structure, Brosey et al. (2015) show that the flexibility and interactions of the modular domains of RPA are altered by ssDNA binding and suggest that these changes in configurational freedom are important for the many functions of RPA.