A multistep damage recognition mechanism for global genomic nucleotide excision repair

Abstract

A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

“…More detailed biochemical analyses with defined nucleic acid substrates revealed that XPC protein displays a general affinity for DNA sites that deviate from the canonical Watson-Crick geometry, including single-stranded loops, mismatched bubbles or single-stranded overhangs [35,36]. According to scanning force microscopy studies, the binding of XPC protein to DNA induces a kink of 39-49º in the nucleic acid backbone regardless of whether the substrate is damaged or not 7 [37].…”

Dynamics of a Benzo[a]pyrene-derived Guanine DNA Lesion in TGT and CGC Sequence Contexts: Enhanced Mobility in TGT Explains Conformational Heterogeneity, Flexible Bending, and Greater Susceptibility to Nucleotide Excision Repair

“…15 According to their bipartite model of NER, the NER machinery first recognizes disruptions of Watson-Crick hydrogen bonding, followed by an unspecified second verification step that ensures that a chemically modified nucleobase is indeed present. 7,8,16,48,49 This has been called an "indirect readout mechanism" by Dip et al 48 Our detailed structural analyses based on MD simulations have previously provided further insights into the nature of DNA distortions that provoke NER susceptibility. [50][51][52][53] We found that a number of other structural parameters, besides Watson-Crick hydrogen bonding, are compromised by bulky lesions, a phenomenon we termed the multipartite mechanism of lesion recognition.…”

“…The basic hypothesis in the field of NER is that the mammalian DNA repair apparatus recognizes structural distortions and thermodynamic destabilization in the DNA duplex caused by the lesions, followed by a verification step that detects the presence of a chemically modified nucleobase and the excision of a fragment 24-32 nucleotides long that contains the damaged base. [3][4][5][6][7][8][9][10][11] It is now established that the first and rate-determining step in NER is the recognition of the bulky lesions by the XPC/HR23B protein heterodimer complex. 12 Since the NER machinery processes a diverse array of bulky lesions with different efficiencies, the conformational features that invoke NER are of great interest.…”

Transcription-coupled nucleotide excision repair in mammalian cells: molecular mechanisms and biological effects

“…The minimal set of proteins required to perform complete GG-NER (more than 30 polypeptides) has been defined using in vitro reconstituted systems [8,9] and specific roles have been assigned to the various factors involved. The DNA damage recognition protein complexes UV-DDB and XPC-RAD23B are required for the efficient recruitment of all of the following NER proteins to the damaged DNA [10][11][12][13]. The basal transcription factor TFIIH has an essential role in NER as two of its components, i.e.…”