Domains in the XPA protein important in its role as a processivity factor

Abstract

XPA is a protein essential for nucleotide excision repair (NER) where it is thought to function in damage recognition/verification. We have proposed an additional role, that of a processivity factor, conferring a processive mechanism of action on XPF and XPG, the endonucleases, involved in NER. The present study was undertaken to examine the domain(s) in the XPA gene that are important for the ability of the XPA protein to function as a processivity factor. Using site-directed mutagenesis, mutations were created in several of the exons of XPA and mutant XPA proteins produced. The results showed that the DNA binding domain of XPA is critical for its ability to act as a processivity factor. Mutations in both the zinc finger motif and the large basic cleft in this domain eliminated the ability of XPA to confer a processive mechanism of action on the endonucleases involved in NER.

Fig. 1

Electrophoretic analysis of the native and mutant recombinant XPA proteins. The purified XPA proteins were electrophoresed on 12% SDS-PAGE and electroblotted onto nitrocellulose: (A) Native, (B) E2 mutant, (C) E5 mutant, and (D) E3 mutant XPA proteins. Lane 1, the membranes were stained with colloidal gold; lane 2, a duplicate membrane was analyzed by western blot analysis using HisProbe-HRP. Arrows indicate the location of the XPA protein. The position of molecular weight markers is indicated.

Fig. 2

Influence of the native and mutant XPA proteins on the mechanism of action utilized by NER endonucleases in XPA cells for locating sites of damage. Endonuclease activity was measured on UVC-irradiated DNA in the presence of a similarly damaged competitor DNA. (A) Chromatin-associated proteins from normal cells were incubated with UVC-irradiated DNA for 40 minutes after which time the UVC-irradiated competitor DNA was added and incubation continued. (B-F) Chromatin-associated proteins from XPA cells plus either native or mutant XPA proteins were incubated with UVC-irradiated DNA for 40 minutes and then the UVC-irradiated competitor DNA was added and incubation continued. The XPA proteins included in the reactions were: (B) native XPA (rXPA); (C) E2 mutant; (D) E3 mutant purified only on a Ni²⁺-NTA column; (E) rXPA purified only on a Ni²⁺-NTA column; (F) E5 mutant. Endonuclease activity was expressed as the number of breaks per DNA molecule. Vertical lines represent ± SEM for 3 to 4 experiments.

Fig. 3

Binding of native and mutant XPA proteins to UVC irradiated DNA. A biotinylated 93 bp DNA substrate was irradiated with UVC light and reacted with native or mutant XPA protein. Streptavidin-coated acrylamide beads were added to the reaction and the bound proteins eluted and analyzed by western blot. Binding of: (A) native XPA protein to: damaged DNA (lane 1), beads alone (lanes 2 and 4), undamaged DNA (lane 3), (B) E2 mutant XPA protein to: damaged DNA (lane 1), beads alone (lane 2), (C) E3 mutant XPA protein to damaged DNA (lane 1) or beads alone (lane 2); native XPA protein purified only on a Ni²⁺-NTA column to damaged DNA (lane 3) or beads alone (lane 4), (D) E5 mutant XPA protein to: damaged DNA (lane 1), beads alone (lane 2).

Fig. 4

A map of the XPA gene and the domains of the XPA protein each encodes. Numbers refer to amino acid numbers for the XPA protein (Adapted from reference 30).