Structural and functional studies of the human ERCC1-XPF DNA repair complex

Abstract

During eukaryotic Nucleotide Excision Repair, DNA cleavage by XPF requires heterodimer formation with ERCC1. The aim of the current thesis was to address the structure-function relationship of the obligate ERCC1/XPF heterodimer. In this line, we have determined the structure of ERCC1/XPF C-terminal interacting domains by NMR. ERCC1 and XPF partners share the same fold, the double helix-hairpin-helix motif (HhH2), and complex in a symmetry related manner. The same dimer interface is observed in the archaeal XPF, which forms homodimers. The archaeal HhH2 domains bind equally to DNA minor grooves, in agreement with the substrate specificity of the archaeal XPF nuclease. In contrast, human ERCC1/XPF cleaves relevant DNA substrates, which consist of one minor groove. We have shown that exclusively the HhH2 domain of ERCC1 mediates minor groove binding. The functional asymmetry in humans is related to the altered second hairpin of XPF, revealed by the structure. Though non-functional this domain acts as scaffold for the folding of the ERCC1 corresponding domain. We concluded that ERCC1 possesses the DNA binding activity that serves to localize the XPF nuclease activity to the NER pre-incision complex and allow for damage excision. Next, we determined the structure of the ERCC1 central domain and investigate its contribution to the heterodimeric function. Although this domain shows structural homology with the catalytically active XPF nuclease domain, it has a distinct function by performing interactions with XPA, another NER protein. This function is crucial as it anchors the XPF nuclease in the pre-incision NER complex and subsequently allows cleavage to occur. Remarkably, the XPA binding by ERCC1 and the catalytic function of XPF are dependent on structurally homologous regions. Furthermore the ERCC1 central domain can bind ss-DNA. XPA and DNA interactions can happen simultaneously through distinct surfaces and indicate the significant dual role of the ERCC1 central domain in targeting the XPF nuclease to the sites of DNA damage. Overall, the structural data from human and archaeal species show that the human ERCC1 and XPF proteins share the architectural subunits of the archaeal XPF homodimer. The structural similarities provide evidence for the common origin of the human proteins. Based on the functional data we proposed that ERCC1 and XPF genes evolved by subfunctionalization, which resulted in the partitioning of the original functions after duplication of the ancestral XPF gene. This evolutionary scenario is able to explain the preservation of the ERCC1 gene and the concomitant transition from homo- to obligate hetero-association in eukaryotes. Structural and biochemical work on the first reported ERCC1 deficiency in humans is on going to understand the mutant ERCC1/XPF dysfunction.