Biophysical characterization of XPA-ERCC1, ATR-ATRIP, and Rad9 in human DNA damage repair pathway

Abstract

In human cells, DNA damage induces two important cellular responses: removal of DNA damage as well as activation of a DNA damage checkpoint. In particular, nucleotide excision repair (NER) is the major pathway for repairing a wide rage of DNA lesions. DNA repair proteins XPA and ERCC1 are well-known to be the absolutely required for DNA-binding and incision steps of NER, respectively. ATR, ATRIP and hRad9 proteins are involved in the DNA damage checkpoints as representative damage sensors. In order to elucidate XPA``s specific role in regulating XPF-ERCC1 5`` endonuclease complex in NER pathway, the specific association between XPA-ERCC 1 interaction should be investigated. Here, the interaction between XPA and ERCC1 has been examined using XPA (58-104) and ERCC1 (1-121) fragments. In vitro binding assays indicate that there is no particular domain-domain interaction between XPA (58-104) and ERCC1 (1-121). In addition, the biophysical characterization data in this study show that the interaction between the identified binding domains of XPA and ERCC1 is too weak to be recognized by NMR technique, possibly due to the extremely fast exchange. Otherwise, the determined binding domains of XPA and ERCC1 are insufficient for the protein-protein interaction. Likewise, XPA (72-84) and ERCC1 (93-120) fragments might not be the minimal binding domains for the mutual interaction as previously reported. Furthermore, this study provides some evidence that both XPA (58-104) and ERCCI (1-121) might be intrinsically unstructured, based on the primary sequence analysis, SDS-PAGE analyses of the purified proteins and thrombin cleavage reactions, and gel filtration chromatography data. Two groups of proteins, ATR-ATRIP complex and hRad9-hHus 1-hRad 1 heterotrimer, have been identified as checkpoint-specific damage sensors to delay cell-cycle progression. To understand the mechanisms of DNA damage sensing, various plasmids harboring individual domains of ATR, ATRIP, and Rad9 were ...