Structural insights into the recognition of cisplatin and AAF-dG lesion by Rad14 (XPA)

Abstract

Nucleotide excision repair (NER) is responsible for the removal of a large variety of structurally diverse DNA lesions. Mutations of the involved proteins cause the xeroderma pigmentosum (XP) cancer predisposition syndrome. Although the general mechanism of the NER process is well studied, the function of the XPA protein, which is of central importance for successful NER, has remained enigmatic. It is known, that XPA binds kinked DNA structures and that it interacts also with DNA duplexes containing certain lesions, but the mechanism of interactions is unknown. Here we present two crystal structures of the DNA binding domain (DBD) of the yeast XPA homolog Rad14 bound to DNA with either a cisplatin lesion (1,2-GG) or an acetylaminofluorene adduct (AAF-dG). In the structures, we see that two Rad14 molecules bind to the duplex, which induces DNA melting of the duplex remote from the lesion. Each monomer interrogates the duplex with a β-hairpin, which creates a 13mer duplex recognition motif additionally characterized by a sharp 70°DNA kink at the position of the lesion. Although the 1,2-GG lesion stabilizes the kink due to the covalent fixation of the crosslinked dG bases at a 90°angle, the AAF-dG fully intercalates into the duplex to stabilize the kinked structure.

XPA: A key scaffold for human nucleotide excision repair

“…Notably, the two structures are nearly identical except for the differences in the lesions, as reflected in the RMSD over all protein atoms of only 0.22 Å. The two key findings from the Rad14 structures are (i) two molecules of Rad14 bind to each side of the lesion-containing duplex, and (ii) the duplexes are kinked by 70° (Figure 3) 45 . The interaction of Rad14 with the ss-dsDNA junction as observed in these structure is consistent with previous studies indicating that human XPA also preferentially binds to junction DNA 26 .…”

“…In 2015, Kisker, Carrell and co-workers reported X-ray crystal structures at 1.8-2.8 Å resolution for the S. cerevisiae XPA homolog Rad14 in complex with damage containing DNA (Figure 3) 45 . These were the first high-resolution 3D structures of an XPA homolog in complex with DNA: one was with duplex DNA containing cisplatin that forms a 1,2-GG intra-strand crosslink (PDB: 5A39) and the second was with the same duplex containing a N -(deoxyguanosin-8-yl)-2-acetylaminofluorene (AAF) (PDB: 5A3D) adduct (Figure 3).…”

“…As noted above, XPA 98-219 has severely reduced DNA-binding activity; a 20 residue C-terminal extension is required to reproduce the DNA binding activity of FL XPA 28 . It is therefore surprising that FL-XPA, FL-Rad14, and Rad14t bind duplex DNA containing cisplatin or AAF lesions very tightly 45 . Moreover, Rad14 does not bind to duplexes containing other commonly studied DNA lesions (e.g.…”

“…Moreover, Rad14 does not bind to duplexes containing other commonly studied DNA lesions (e.g. (6-4)photoproduct ((6-4)PP), cyclobutane pyrimidine dimer (CPD)) with appreciable affinity 45 . In light of these observations, it would be interesting to know if XPA 98-219 binds these substrates with comparable affinity.…”

Chromatin Regulates Genome Targeting with Cisplatin

“…[21] It is also conceivable that HDAC inhibition promoted lesion bypass at the expense of DNAr epair, [22] thereby leading to detectable clusters of DNA-Pt prone to trigger apoptosis through other mechanisms.…”