The ultraviolet (UV) radiation-induced DNA lesions play a causal role in many prevalent genetic skin-related diseases and cancers. The damage sensing protein Rad4/XPC specifically recognizes and repairs these lesions with high fidelity and safeguards genome integrity. Despite considerable progress, the mechanistic details of the mode of action of Rad4/XPC in damage recognition remain obscure. The present study investigates the mechanism, energetics, dynamics, and the molecular basis for the sequence specificity of mismatch recognition by Rad4/XPC. We dissect the following three key molecular events that occur as Rad4/XPC tries to recognize and bind to DNA lesions/mismatches: (a) the association of Rad4/XPC with the damaged/mismatched DNA, (b) the insertion of a lesion-sensing β-hairpin of Rad4/XPC into the damage/mismatch site and (c) the flipping of a pair of nucleotide bases at the damage/mismatch site. Using suitable reaction coordinates, the free energy surfaces for these events are determined using molecular dynamics (MD) and umbrella sampling simulations on three mismatched (CCC/CCC, TTT/TTT and TAT/TAT mismatches) Rad4-DNA complexes. The study identifies the key determinants of the sequence-dependent specificity of Rad4 for the mismatches and explores the ramifications of specificity in the aforementioned events. The results unravel the molecular basis for the high specificity of Rad4 towards CCC/CCC mismatch and lower specificity for the TAT/TAT mismatch. A strong correlation between the depth of β-hairpin insertion into the DNA duplex and the degree of coupling between the hairpin insertion and the flipping of bases is also observed. The interplay of the conformational flexibility of mismatched bases, the depth of β-hairpin insertion, Rad4-DNA association energetics and the Rad4 specificity explored here complement recent experimental FRET studies on Rad4-DNA complexes.

The homodimeric catabolite activator protein (CAP) regulates the transcription of several bacterial genes based on the cellular concentration of cyclic adenosine monophosphate (cAMP). The binding of cAMP to CAP triggers allosteric communication between the cAMP binding domains (CBD) and DNA binding domains (DBD) of CAP, which entails repositioning of DNA recognition helices (F-helices) in the DBD to dock favorably to the target DNA. Despite considerable progress, much remains to be understood about the mechanistic details of DNA recognition by CAP and about the map of allosteric pathways involved in CAP-mediated gene transcription. The present study uses molecular dynamics and umbrella sampling simulations to investigate the mechanism of cAMP-and DNAinduced changes in the conformation and energetics of F-helices observed during the allosteric regulation of CAP by cAMP and the subsequent binding to the DNA promoter region. Using novel collective variables, the free energy profiles associated with the orientation and dynamics of F-helices in the unliganded, cAMP-bound, and cAMP-DNA-bound states of CAP are calculated and compared. The binding-induced alterations in the resultant free energy profiles reveal important flexibility constraints imposed on DBD upon cAMP and DNA binding. A comprehensive analysis of residue-wise interaction maps reveals potential allosteric pathways between CBD and DBD that facilitate the allosteric transduction of regulatory signals in CAP. The revelation that the predicted allosteric pathways crisscross the intersubunit interface offers important clues on the microscopic origin of the intersubunit cooperativity and dimer stability of CAP.