Sequential Assembly of the Nucleotide Excision Repair Factors In Vivo (original) (raw)

Leiden University Medical Center ema et al., 1992). Examination of the repair kinetics of Wassenaarseweg 72 structurally different DNA lesions has disclosed that 2333 AL, Leiden TCR functions as an efficient repair system for transcrip-2 Swammerdam Institute for Life Sciences tion-blocking lesions that are poorly repaired by GGR BioCentrum Amsterdam (Hanawalt, 1995). In the case of UV-induced photole-University of Amsterdam sions, repair of 6-4PP is fast throughout the genome Plantage Muidergracht 12 and is dominated by GGR. In contrast, GGR of CPD is 1018 TV, Amsterdam relatively slow, but TCR causes accelerated removal of 3 Department of Cell Biology and Genetics this lesion from the transcribed strand of expressed Medical Genetics Center genes (van Hoffen et al., 1995). Erasmus University Rotterdam Cell fusion experiments have revealed seven XP ge-PO Box 1738 netic complementation groups (XP-A through XP-G) that 3000 DR, Rotterdam represent different proteins in the NER pathway. Among The Netherlands the various complementation groups, XP-C is unique, as only GGR is compromised in this group (Venema et al., 1991). The photosensitive inherited disorder Cock-Summary ayne syndrome (CS), on the other hand, is associated with defective TCR, while GGR is unaffected (van Hoffen Here, we describe the assembly of the nucleotide exciet al., 1993; Venema et al., 1990). At the cellular level, sion repair (NER) complex in normal and repair-defithe two CS genetic complementation groups (CS-A and cient (xeroderma pigmentosum) human cells, em-CS-B) are characterized by a lack of recovery of inhibited ploying a novel technique of local UV irradiation RNA synthesis following exposure to DNA damaging combined with fluorescent antibody labeling. The agents, a phenomenon that has been related to defecdamage recognition complex XPC-hHR23B appears tive TCR (Mayne and Lehmann, 1982; Venema et al., to be essential for the recruitment of all subsequent 1990). NER factors in the preincision complex, including tran-Incision of damaged DNA is a multistep process inscription repair factor TFIIH. XPA associates relatively volving recognition of the DNA damage followed by late, is required for anchoring of ERCC1-XPF, and may opening up of the DNA helix around the lesion, dual be essential for activation of the endonuclease activity incision, and subsequent excision of the oligonucleotide of XPG. These findings identify XPC as the earliest containing the DNA lesion (de Laat et al., 1999). From known NER factor in the reaction mechanism, give in vitro biochemistry, it is not clear in which order various insight into the order of subsequent NER components, NER factors act in the reaction mechanism, particularly provide evidence for a dual role of XPA, and support with respect to the first stages including the crucial a concept of sequential assembly of repair proteins damage recognition step. In addition, it is not evident at the site of the damage rather than a preassembled what the organization of repair is in vivo-are NER facrepairosome. tors preassembled in a NER holocomplex, in distinct subassemblies, or as individual factors that transiently Introduction interact at the site of the lesion? The identity of the damage recognition factor has been a matter of debate. In eukaryotes, nucleotide excision repair (NER) is a ver-Several putative candidates have been proposed, insatile and highly conserved repair system capable of cluding the XPA-replication protein A (RPA) complex removing a wide range of DNA lesions that distort the (Asahina et al., 1994; Li et al., 1995b), the XPC-hHR23B stacking of the DNA double helix, including the shortcomplex (Reardon et al., 1996, Batty and Wood, 2000; wave ultraviolet (UV) light-induced cyclobutane pyrimi-Yokoi et al., 2000), and the p48-p127 complex, also dine dimers (CPD) and 6-4 photoproducts (6-4PP). In termed damaged DNA binding (DDB) protein (Chu and humans, repair of UV-induced photolesions is entirely Chang, 1988; Tang et al., 2000). DDB has not been implidependent on NER, and mutations in NER proteins have cated so far in the damage recognition step in in vitro been associated with the inherited disorder xeroderma experiments. Results obtained in in vitro experiments pigmentosum (XP) (Bootsma et al., 1998). XP patients are contradictory as to whether XPC-hHR23B or XPA-RPA is the principal damage recognition protein. Findings by Sugasawa and coworkers using N-acetoxy-2-4 Correspondence: zeeland@lumc.nl