Influence of centrin 2 on the interaction of nucleotide excision repair factors with damaged DNA

Abstract

We have examined the influence of centrin 2 (Cen2) on the interaction of nucleotide excision repair factors (XPC-HR23b, RPA, and XPA) with 48-mer DNA duplexes bearing the dUMP derivative 5-{3-[6-(carboxyamidofluoresceinyl)amidocapromoyl]allyl}-2'-deoxyuridine-5'-monophosphate. The fluorescein residue linked to the nucleotide base imitates a bulky lesion of DNA. Cen2 stimulated the binding and increased the yield of DNA adducts with XPC-HR23b, a protein recognizing bulky damages in DNA. Stimulation of the binding was most pronounced in the presence of Mg(2+) and demonstrated a bell-shaped dependence on Cen2 concentration. The addition of Cen2 changed the stoichiometry of RPA-DNA complexes and diminished the yield of RPA-DNA covalent crosslinks. We have shown that Cen2 influences the binding of RPA and XPA with DNA, which results in formation of additional DNA-protein complexes possibly including Cen2. We have also found some evidence of direct contacts between Cen2 and DNA. These results in concert with the literature data suggest that Cen2 can be a regulatory element in the nucleotide excision repair system.

XPA: A key scaffold for human nucleotide excision repair

“…XPC functions as a heterotrimer with HR23B and centrin-2, which stimulate XPC DNA binding activity and increases cellular stability 49 . Once engaged on the damaged duplex, XPC recruits the TFIIH complex 43 .…”

“…XPA (and RPA) was originally thought to contribute to damage recognition and verification, in part due to its interaction with XPC. However, more recent experiments showed that XPA (in concert with RPA) is recruited to the damaged site after the formation of the NER bubble 49 .…”

Calcium-Binding Capacity of Centrin2 Is Required for Linear POC5 Assembly but Not for Nucleotide Excision Repair

“…Centrin2 co-fractionates with xeroderma pigmentosum group C protein (XPC) and its interactor, HRAD23B, key proteins that direct the initial DNA damage recognition step of an NER pathway termed global genome repair [31,32]. The role played by centrin2 in NER is unclear: in vitro NER can be carried out in its absence, although it increases NER activity somewhat [32–35]. Nevertheless, centrin2 moves to the nucleus after UV irradiation in an XPC-dependent manner and is required for efficient repair of UV-induced DNA lesions in both human and chicken cells [25,36].…”

A Human XPC Protein Interactome—A Resource

“…XPC functions in GG-NER within the XPC-RAD23B-CETN2 complex; the interaction of XPC and RAD23B has been shown to increase the affinity of XPC for damaged DNA [8] while the interaction between XPC and CETN2 has been shown to stabilize XPC and promote NER [1,9]. The interactions of XPC with CETN2, RAD23B, and XPA have been biochemically characterized [10,11].…”