Sequence specificity, energetics and mechanism of mismatch recognition by DNA damage sensing protein Rad4/XPC

Abstract

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.

Xeroderma Pigmentosum Complementation Group C (XPC): Emerging Roles in Non-Dermatologic Malignancies

“…Recent in vitro studies have elucidated a role of Rad4, the yeast homolog of XPC, in the recognition and repair of multiple contiguous mismatched base pairs ( 29 ). Specifically, in vitro binding and conformational studies suggest that Rad4/XPC interacts with the nucleotides directly across from the mismatched bases (on the complementary strand), leading to subsequent unwinding, DNA bending, and flipping out of the mismatched nucleotides and stabilization of this conformation to allow for subsequent DNA repair ( 29 , 30 ). These studies suggest a mechanism by which XPC acts as a universal DNA damage sensor, recognizing sites of DNA distortion and binding in a lesion-agnostic fashion (“non-specific binding”).…”

“…These studies suggest a mechanism by which XPC acts as a universal DNA damage sensor, recognizing sites of DNA distortion and binding in a lesion-agnostic fashion (“non-specific binding”). Indeed, recent studies suggest that the Rad4/XPC-DNA binding leads to different conformational changes based on the lesion type, such that XPC bound at the site of UV-induced DNA damage (“specific binding”) facilitates recruitment and initiation of NER while “non-specific” binding to minimally strand-distorting lesions facilitates non-NER repair ( 29 , 30 ). Extensive structural analysis has been done to understand sequence and structural changes of DNA lesions sensitive and resistant to Rad4/XPC binding and subsequent GG-NER efficiency ( 31 ).…”

Formation and Recognition of UV-Induced DNA Damage within Genome Complexity

“…To stably adopt the bound conformation, the XPC complex needs to overcome a consequent energy barrier, which was described as a primary regulator for the recognition specificity [ 129 , 130 ]. Indeed, DNA damages induce structural changes of the DNA helix structure and weak base pairing, leading to a decrease of the energy barrier that the recognition complex needs to overcome for efficient binding [ 131 ]. In other words, XPC may patrol along the DNA until encountering a disturbed helical structure with weak base pairing, allowing XPC to stably bind the lesion site [ 128 , 129 , 130 , 131 , 132 ].…”

“…Indeed, DNA damages induce structural changes of the DNA helix structure and weak base pairing, leading to a decrease of the energy barrier that the recognition complex needs to overcome for efficient binding [ 131 ]. In other words, XPC may patrol along the DNA until encountering a disturbed helical structure with weak base pairing, allowing XPC to stably bind the lesion site [ 128 , 129 , 130 , 131 , 132 ]. This process likely explains how the XPC complex detects several different types DNA damages and why the identification of particular lesions is more efficient.…”

XPC multifaceted roles beyond DNA damage repair: p53-dependent and p53-independent functions of XPC in cell fate decisions

“…In a recent computational study, Panigrahi et al used molecular dynamics and umbrella sampling simulations to investigate mismatch recognition by Rad4/XPC. The dynamic and energetic characterization of the order and extent of specificity of Rad4/XPC for 3-bp of mismatched sequences demonstrated that Rad4 is highly specific to a mismatch of CCC/CCC, while it recognizes a TTT/TTT mismatch with intermediate specificity and only poorly recognizes TAT/TAT mismatch [24]. Another study showed that XPC binding affinity to DNA bulky lesions is lost when the single nucleotide in the complementary strand opposite the lesion is deleted [25].…”