Cloning, expression and purification of human homologs XPB and XPD from hyperthermophilic Archaeon, Pyrococcus abyssi

Project leader: Milan Škorvaga
Project duration: 2006 - 2008

The importance of DNA repair pathways for human health is demonstrated by the existence of several genetic disorders associated with defects in DNA repair or responses to DNA damage . The nucleotide excision repair (NER) pathway is unique in its capability to recognize and repair a large variety of structurally unrelated DNA lesions. Major challenge is to understand the molecular mechanisms of damage recognition process which is impossible without structural information and structure-function analysis of the proteins involved. We propose to carry out these studies on human homologs of XPB and XPD helicase from hyperthermophilic Archaeon, Pyrococcus abyssi (Pab). We wish to clone wild-type Pab XPB and XPD as well as to construct point mutants found in selected XP-D patients, and overproduce them for crystallization. Structural analysis of these proteins may help understand the molecular mechanisms of action of respective DNA helicases during NER as well as the nature of the respective defect in patient and possibly make a progress in targeting the therapy.