Kaoru Sugasawa

*Biosignal Research Center, Organization of Advanced Science and Technology Kobe University Japan* 

### **1. Introduction**

452 DNA Repair

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homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA

(2006). sbcB15 And DeltasbcB mutations activate two types of recf recombination

As a blueprint for genetic information, the structural and functional integrity of DNA must be maintained during cell division and gamete formation. However, this fundamental principle is threatened continuously by the vulnerability of DNA itself and/or by assaults from endogenously produced agents, such as reactive oxygen species and other metabolites, as well as various environmental agents including ultraviolet light (UV), ionizing radiation and chemical compounds (Friedberg et al., 2006). Among the DNA components, bases in particular are frequently the targets for such insults. Because DNA replication and transcription rely on the formation of specific base pairs, even a subtle change in the base structures can compromise faithful propagation and the expression of genetic information. For instance, replicative DNA polymerases, which exhibit very high intrinsic fidelity, are often blocked at sites where template bases are modified, which can lead to replication fork collapse and consequent chromosomal aberrations and/or cell death. This problem is overcome, at least partly, by translesion DNA synthesis, which is an error-prone process (Friedberg et al., 2005). To minimize the risk of mutagenesis, it is crucial for growing cells to detect and to remove damaged bases as much as possible before replication forks collide with them.

Nucleotide excision repair (NER) is a major DNA repair pathway that can eliminate an extremely broad spectrum of base damage. The NER substrates include dipyrimidinic UV photolesions, such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PPs), intrastrand crosslinks caused by bifunctional alkylating agents (e.g., cisplatin), and bulky base adducts induced by numerous chemical carcinogens (Gillet & Schärer, 2006). The common feature shared by all of these insults does not reside in their chemical structure, but rather in the accompanying distortions of the otherwise regular DNA helical structure. Two subpathways are associated with mammalian NER: global genome NER (GG-NER) and transcription-coupled NER (TC-NER). GG-NER is a general pathway that operates throughout the genome. It minimizes the collision of replication forks with damaged bases and, thereby, contributes to the maintenance of genome integrity (Gillet & Schärer, 2006). TC-NER is specialized to remove transcription-blocking lesions from the template DNA strands, which ensures rapid recovery of transcriptional activity and thus averts apoptosis (Hanawalt & Spivak, 2008). In humans, hereditary defects in NER are associated with several autosomal recessive disorders, including xeroderma

DNA Damage Recognition for Mammalian Global Genome Nucleotide Excision Repair 455

One of the remarkable characteristics of GG-NER resides in its extremely broad substrate specificity, which encompasses UV-induced photolesions and other bulky base adducts that can be induced by numerous chemical compounds (Gillet & Schärer, 2006). These GG-NER substrates are associated with considerable levels of DNA helical distortion. This situation is in marked contrast to substrates for base excision repair (BER), such as uracils and oxidative base lesions, which are supposed to induce only marginal structural distortions. Initial damage detection for BER is accomplished by a set of DNA glycosylases, each of which exhibits a certain (partially overlapping) range of substrate specificity. In contrast, a virtually infinite spectrum of helix-distorting insults can be handled by the unified molecular machinery in GG-NER. In addition, GG-NER must survey the huge genome continuously and discriminate a small number of injured bases from normal bases with very high efficiency and accuracy. Recent biochemical studies have uncovered some of the

The *XPC* gene was isolated from a cDNA expression library (Legerski & Peterson, 1992) that corrected the UV sensitivity of fibroblasts from patients with XP-C. Cells lacking XPC are incompetent for GG-NER, but TC-NER functions normally (Venema et al., 1990). By using the cell-free NER system, a protein factor that is missing in XP-C cells was purified from HeLa cell extracts (Masutani et al., 1994; Shivji et al., 1994). This biochemical approach revealed that the XPC protein forms a stable complex in vivo with 1 of the 2 human homologues of *Saccharomyces cerevisiae* Rad23p (designated RAD23A and RAD23B). Depletion of RAD23 markedly destabilized the XPC protein, thereby compromising GG-NER function (Ng et al., 2003; Okuda et al., 2004). Another component of the XPC complex, centrin-2 (Araki et al., 2001; Nishi et al., 2005), belongs to the calmodulin superfamily of small calcium-binding proteins containing 4 conserved EF-hand motifs. A subpopulation of centrin-2 localizes to the centrosomes and plays a vital role in cell cycle regulation (Lutz et al., 2001; Salisbury et al., 2002). Centrin-2 also binds to an α-helix near the C-terminus of XPC: this interaction potentiates the DNA-binding activity of the complex (Bunick et al.,

The XPC protein complex has been known to be associated with DNA-binding activity since it was first purified (Masutani et al., 1994; Shivji et al., 1994), although its preference for damaged DNA was discovered sometime later (Batty et al., 2000; Sugasawa et al., 1998). With conventional electrophoretic mobility shift assays (EMSAs) and DNase I footprint analyses with defined DNA substrates, we demonstrated that XPC prefers to associate with sites containing a helix-distorting lesion, such as 6-4PP or *N*-(guanin-8-yl) *N*-acetyl-2-aminofluorene (dG-AAF) adduct (Sugasawa et al., 1998; Sugasawa et al., 2001). However, the addition of an appropriate competitor DNA was necessary to reveal the damage specificity, by preventing XPC from binding to the undamaged part of the DNA. Several physicochemical approaches subsequently were undertaken to assess the affinities of XPC for various DNA structures in more dynamic states (Hey et al., 2002; Roche et al., 2008;

Involvement of the XPC complex in the very early stages of NER was first proposed on the basis of the results obtained with the cell-free NER system (Sugasawa et al., 1998). In this system, 2 plasmid DNAs containing AAF adducts were preincubated separately with

**2. Primary damage sensors for the initiation of GG-NER** 

sophisticated molecular mechanisms that achieve this difficult task.

2006; Nishi et al., 2005; Popescu et al., 2003; Thompson et al., 2006).

**2.1 Indirect sensing of DNA damage by XPC** 

Trego & Turchi, 2006).

pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD) (Bootsma et al., 2001). The clinical hallmarks exhibited by patients with XP, which include marked photosensitivity and a predisposition to skin cancer, explicitly indicate that the impaired repair of UV-induced DNA photolesions promotes mutagenesis and carcinogenesis in the skin. Numerous genetic complementation groups have been identified for the above diseases, including 8 for XP (XP-A through -G, and variant), 2 for CS (CS-A and -B) and 1 for TTD (TTD-A). Cloning of the responsible genes has revealed that all of them encode proteins involved in the NER pathway. The notable exception is the XP variant (*XPV*) gene encoding DNA polymerase η that is involved in translesion DNA synthesis, but not in NER. Another important milestone in elucidating the NER mechanism has been the establishment of the cell-free system, which faithfully recapitulates the NER reaction with human whole cell extracts. Together, genetic and biochemical studies have successfully identified more than 30 polypeptides that are involved in mammalian GG-NER (Fig. 1). A fundamental challenge for GG-NER is that the cells must detect a small number of injured bases among the vast excess of normal bases comprising the huge genome. Although the complete network of mechanisms has not yet been entirely uncovered, recent studies have revealed some of the sophisticated molecular mechanisms that accomplish this difficult task, which involves concerted actions of multiple protein factors. This chapter overviews the latest progress in our understanding of the damage-recognition mechanism for mammalian GG-NER.

Fig. 1. Model of the mammalian NER mechanism. The 2 subpathways, GG-NER and TC-NER, differ in their strategies for initial damage recognition, but converge into a common process. The disease-related gene products are indicated by letters in different colors: XP, red; CS, green; TTD, blue.
