Escherichia coli dinD is an SOS gene up-regulated in response to DNA damage. We find that the purified DinD protein is a novel inhibitor of RecA-mediated DNA strand exchange activities. Most modulators of RecA protein activity act by controlling the amount of RecA protein bound to single-stranded DNA by affecting either the loading of RecA protein onto DNA or the disassembly of RecA nucleoprotein filaments bound to singlestranded DNA. The DinD protein, however, acts postsynaptically to inhibit RecA during an on-going DNA strand exchange, likely through the disassembly of RecA filaments. DinD protein does not affect RecA single-stranded DNA filaments but efficiently disassembles RecA when bound to two or more DNA strands, effectively halting RecA-mediated branch migration. By utilizing a nonspecific duplex DNA-binding protein, YebG, we show that the DinD effect is not simply due to duplex DNA sequestration. We present a model suggesting that the negative effects of DinD protein are targeted to a specific conformational state of the RecA protein and discuss the potential role of DinD protein in the regulation of recombinational DNA repair.The Escherichia coli SOS response is a coordinately regulated network of genes induced in reaction to heavy or persistent DNA damage (1). The SOS regulon is repressed by the LexA protein, which is inactivated as a repressor upon cellular stress such as heavy DNA damage. The cellular signal for SOS induction is the RecA protein bound to single-stranded DNA (ssDNA). 2RecA is the central DNA recombinase in bacteria and carries out several distinct functions when activated (2). The most appreciated function of RecA is the catalysis of homologous DNA recombination crucial to the generation of genetic diversity. However, based on frequency of use, the primary role of RecA lies in the multiple pathways for the recombinational repair of stalled DNA replication forks. Through much in vitro work, it has become increasingly apparent that the RecA protein is under considerable control by a network of proteins that function to modulate when and where RecA protein binds to DNA (3). The PsiB protein has recently been shown to bind to free RecA protein, effectively inhibiting RecA from nucleating onto ssDNA (4). RecA is also inhibited from nucleating onto ssDNA bound by the single-stranded DNA-binding protein (SSB) (5). The SSB-imposed inhibition is relieved by the action of the RecF, RecO, and RecR proteins (3). Following nucleation, RecA protein protomers assemble into a nucleoprotein filament, extending cooperatively in the 5Ј to 3Ј direction. The RdgC protein can inhibit RecA binding to ssDNA and can interfere with homologous DNA pairing by binding to duplex DNA (6, 7). Some proteins shown to dismantle RecA filaments are known DNA translocases, such as UvrD (8) and PcrA helicases (9). Filament extension can be blocked through the action of the RecX protein (10), whereas the DinI protein antagonizes the function of RecX by stabilizing RecA filaments, inhibiting filament end-dependent disassem...

Interactions of intrinsically disordered proteins (IDP) with their binding partners often involve coupled binding and folding. A long-standing question is the extent to which folding of the IDP is mediated by selection of a folded conformer from the disordered state ensemble rather than folding induced by interaction with the binding partner. Answering this question requires detailed information about the disordered state ensemble, in particular the extent to which the IDP possesses residual structure. Yet obtaining this type of information at near-atomic resolution remains challenging. To address this need, we have developed an approach based on millisecond quench-flow amide H/D exchange and mass spectrometry to measure residual structure. In the present work, we examine residual structure in the disordered CBP-binding domain of ACTR as a model system for validation. Following millisecond H/D exchange and acid quench, digestion with pepsin produced a set of 67 highly-overlapping fragments covering the entire 77-resdidue sequence. Residue-by-residue analysis of empirically-determined H/D exchange halflife obtained from each ACTR fragment provided exchange kinetics at nearresidue resolution. In ACTR, we found that the regions that are known adopt an a-helical fold upon binding to CBP became more protected from H/D exchange than the structured loop regions. We also found that most of the N-terminal region, which does not appear in the solved structure, was the least protected. There was also evidence of slight protection in a short stretch of the N-terminal region. Our results are consistent both with a recent analysis of residual structure obtained from NMR secondary shift measurements and with the AGADIR helicity prediction algorithm. Our results demonstrate the utility of millisecond H/D exchange for mapping secondary structural propensity in disordered state ensembles with near-residue resolution.