Replication protein A (RPA) binds to single-stranded DNA (ssDNA) and interacts with over three dozen enzymes and serves as a recruitment hub to coordinate most DNA metabolic processes. RPA binds ssDNA utilizing multiple oligosaccharide/oligonucleotide binding domains and based on their individual DNA binding affinities are classified as high versus low-affinity DNA-binding domains (DBDs). However, recent evidence suggests that the DNA-binding dynamics of DBDs better define their roles. Utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS), we assessed the ssDNA-driven dynamics of the individual domains of human RPA. As expected, ssDNA binding shows HDX changes in DBDs A, B, C, D and E. However, DBD-A and DBD-B are dynamic and do not show robust DNA-dependent protection. DBD-C displays the most extensive changes in HDX, suggesting a major role in stabilizing RPA on ssDNA. Slower allosteric changes transpire in the protein-protein interaction domains and linker regions, and thus do not directly interact with ssDNA. Within a dynamics-based model for RPA, we propose that DBD-A and -B act as the dynamic half and DBD-C, -D and -E function as the less-dynamic half. Thus, segments of ssDNA buried under the dynamic half are likely more readily accessible to RPA-interacting proteins.

The DNA damage checkpoint induces many cellular changes to cope with genotoxic stress. However, persistent checkpoint signaling can be detrimental to growth partly due to blockage of cell cycle resumption. Checkpoint dampening is essential to counter such harmful effects, but its mechanisms remain to be understood. Here, we show that the DNA helicase Srs2 removes a key checkpoint sensor complex, RPA, from chromatin to down-regulate checkpoint signaling in budding yeast. The Srs2 and RPA antagonism is supported by their numerous suppressive genetic interactions. Importantly, moderate reduction of RPA binding to single-strand DNA (ssDNA) rescues hypercheckpoint signaling caused by the loss of Srs2 or its helicase activity. This rescue correlates with a reduction in the accumulated RPA and the associated checkpoint kinase on chromatin in mutants. Moreover, our data suggest that Srs2 regulation of RPA is separable from its roles in recombinational repair and critically contributes to genotoxin resistance. We conclude that dampening checkpoint by Srs2-mediated RPA recycling from chromatin aids cellular survival of genotoxic stress and has potential implications in other types of DNA transactions.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes are poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyper-active enzyme complex, suggesting a role for the N-terminus in auto-inhibition. Hydrogen deuterium exchange mass spectrometry shows that ATP-binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the inter-subunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

The Mediator complex controls transcription of most eukaryotic genes with individual subunits required for the control of particular gene regulons in response to various perturbations. In this study, we reveal the roles of the plant Mediator subunits MED16, MED14, and MED2 in regulating transcription in response to the phytohormone abscisic acid (ABA) and we determine which elements are under their control. Using synthetic promoter reporters we established an effective system for testing relationships between subunits and specific acting motifs in protoplasts. Our results demonstrate that MED16, MED14, and MED2 are required for the full transcriptional activation by ABA of promoters containing both the ABRE (ABA-responsive element) and DRE (drought-responsive element). Using synthetic promoter motif concatamers, we showed that ABA-responsive activation of the ABRE but not the DRE motif was dependent on these three Mediator subunits. Furthermore, the three subunits were required for the control of water loss from leaves but played no role in ABA-dependent growth inhibition, highlighting specificity in their functions. Our results identify new roles for three Mediator subunits, provide a direct demonstration of their function and highlight that our experimental approach can be utilized to identify the function of subunits of plant transcriptional regulators.

Replication protein A (RPA) is an essential single-stranded DNA (ssDNA)-binding protein that sequesters ssDNA and protects it from nucleolytic degradation. The RPA-ssDNA nucleoprotein acts as a hub to recruit over two dozen DNA metabolic enzymes onto ssDNA to coordinate DNA replication, repair, and recombination. RPA functions as a heterotrimer composed of RPA70, RPA32, and RPA14 subunits and has multiple DNA-binding and protein-interaction domains. Several of these domains are connected by disordered linkers allowing RPA to adopt a wide variety of conformations on ssDNA. Here we describe a fluorescence-based tool to monitor the dynamics of select DNA-binding domains of RPA. Noncanonical amino acids are utilized to site-specifically engineer fluorescent probes in Saccharomyces cerevisiae RPA heterologously expressed in BL21 (DE3) and its derivatives. A procedure to synthesize 4-azido-L-phenylalanine (4AZP), a noncanonical amino acid, is also described. Sites for fluorophore positioning that produce a measurable change in fluorescence upon binding to ssDNA are detailed. This fluorescence enhancement through noncanonical amino acid (FEncAA) approach can also be applied to other DNA-binding proteins to investigate the dynamics of protein-nucleic acid interactions.