RESUMO
The XPD protein is a vital subunit of the general transcription factor TFIIH which is not only involved in transcription but is also an essential component of the eukaryotic nucleotide excision DNA repair (NER) pathway. XPD is a superfamily-2 5'-3' helicase containing an iron-sulphur cluster. Its helicase activity is indispensable for NER and it plays a role in the damage verification process. Here, we report the first structure of XPD from Thermoplasma acidophilum (taXPD) in complex with a short DNA fragment, thus revealing the polarity of the translocated strand and providing insights into how the enzyme achieves its 5'-3' directionality. Accompanied by a detailed mutational and biochemical analysis of taXPD, we define the path of the translocated DNA strand through the protein and identify amino acids that are critical for protein function.
Assuntos
Proteínas Arqueais/química , DNA Helicases/química , Thermoplasma/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Helicases/metabolismo , Reparo do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.
Assuntos
Proteínas Arqueais/química , Proteínas Ferro-Enxofre/química , Proteína Grupo D do Xeroderma Pigmentoso/química , Sequência de Aminoácidos , Animais , Proteínas Arqueais/genética , Sequência de Bases , Cristalografia por Raios X , Primers do DNA , Reparo do DNA , Humanos , Proteínas Ferro-Enxofre/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Proteína Grupo D do Xeroderma Pigmentoso/genéticaRESUMO
Xeroderma pigmentosum complementation group D protein (XPD) is an iron-sulfur cluster containing 5'-3' helicase and, in humans, part of the transcription factor TFIIH. TFIIH is involved in nucleotide excision repair as well as in transcription initiation. Recently, three different groups have reported the structures of archaeal XPDs. All structures revealed a four-domain organization with two RecA-like domains, an Arch domain and an iron-sulfur cluster domain. It was possible to rationalize several of the mutations in the human XPD gene that lead to one of the three severe diseases xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy. The different structures are compared and disease-related mutations are discussed.