**3.3 Possible roles for XPA and RPA**

464 DNA Repair

related helicase, exhibits the opposite (3' to 5') polarity, it has been proposed that XPB and XPD may be loaded onto different DNA strands and may move toward the same direction (Dip et al., 2004). This process would enable the simultaneous inspection of both strands, so that discrimination between damaged and undamaged strands can be made depending on which helicase is blocked. However, our results strongly suggest that only 1 strand is subjected to scanning, such that lesions on the other strand, if any, are ignored. Recent mutational analyses have revealed that the ATPase, but not the helicase, activity of XPB is required for NER (Coin et al., 2007): this finding implies that XPB may not mediate the

Fig. 4. Polarity of the XPC binding regulates which DNA strand is scanned by the XPD helicase. For successful loading of XPD onto the damaged strand, XPC must interact with

Another point made by this study was the importance of the XPC binding polarity. As demonstrated by the aforementioned biochemical and structural studies (Min & Pavletich, 2007; Sugasawa et al., 2002), XPC binds to a site containing unpaired bases in an asymmetric fashion. This binding polarity can be controlled intentionally by using a loop structure, in which only 1 DNA strand has unpaired bases. When a loop with either polarity was substituted for a bubble positioned on the 5' side of the CPD, incision at the lesion site was stimulated only by a looped-out sequence in the "undamaged" (CPD-free) DNA strand. In the case where both the loop and CPD were present in the same strand, incision was completely blocked (Sugasawa et al., 2009). These findings strongly suggest that, after XPC interacts with unpaired bases in 1 DNA strand, the XPD helicase in subsequently recruited TFIIH may be loaded onto the other strand and may start scanning in the 5' to 3' direction. According to this model, XPD would be forced to bind the undamaged strand erroneously,

Although this model was deduced from the results of in vitro experiments using rather artificial DNA substrates, it might also apply to normal NER reactions, in which unpaired bases and chemical modifications coexist in close proximity. To induce productive NER, XPC must interact with unpaired bases opposite the lesion, so that the XPD helicase can be loaded successfully onto the damaged strand immediately on the 5' side of the lesion. Intriguingly, with DNA containing a bulky lesion (such as the dG-AAF adduct), XPC exhibits a propensity to bind in a correct orientation in the absence of other factors, most likely because of steric effects preventing interactions between XPC and the modified base (Sugasawa et al., 2009). On the other hand, footprints of XPC on a 6-4PP appear rather

opening of the DNA duplex or movement along a DNA strand.

the undamaged strand.

if the damage-containing strand is looped out.

*XPA*, which was the first cloned XP gene (Tanaka et al., 1990), complemented UV sensitivity of fibroblasts from patients with XP-A. Cultured cells lacking expression of functional *XPA* are defective in both GG-NER and TC-NER, and show extreme sensitivity to killing by UV. The *XPA* gene product is a relatively small protein that is essential for in vitro NER. It shows a DNA-binding activity with a significant preference for various types of damaged DNA (Asahina et al., 1994; Jones & Wood, 1993).

Replication protein A (RPA) is a heterotrimeric protein complex exhibiting remarkable single-stranded DNA-binding activity. RPA is supposed to promote the unwinding of the DNA duplex, stabilize the single-stranded conformation, and stimulate various enzymatic activities, such as DNA polymerases. As the eukaryotic counterpart of bacterial SSB, RPA has been implicated in various DNA metabolisms, including replication, repair, and recombination (Wold, 1997). Its involvement in NER was demonstrated by fractionation and reconstitution of human cell-free extracts used for in vitro NER (Coverley et al., 1991). RPA also binds damaged DNA with significant specificity (Burns et al., 1996; Clugston et al., 1992; He et al., 1995), and the reported interaction between XPA and RPA seems to enhance their damage-specific DNA-binding activities (Buschta-Hedayat et al., 1999; He et al., 1995; Li et al., 1995; Wakasugi & Sancar, 1999).

Although the above findings suggest that the XPA-RPA complex could be responsible for initial damage recognition, the observed specificity and affinity of this complex for damaged DNA seem less pronounced than those of XPC or UV-DDB. In addition, accumulating evidence from biochemical and cell biological studies has supported the conclusion that these factors are more likely to be involved in later stages of the NER process. Both XPA and RPA are essential for the assembly of the NER preincision intermediate complex that contains the fully opened DNA duplex. RPA likely stabilizes the single-stranded conformation of DNA and protects the undamaged strand specifically, while XPA binds around the end of the unwound region on the 5' side of the lesion (Krasikova et al., 2010). Considering the reported physical interactions with a number of NER factors, one of the roles for these factors may be orchestrating the assembly of the preincision complex and correctly arranging other factors, including the 2 incision endonucleases, ERCC1-XPF and XPG.

The XPA protein possesses a zinc-finger domain, which NMR studies revealed is involved in the interaction with RPA. In contrast, its DNA-binding functionality was assigned to a different domain in the protein (Buchko et al., 1998; Buchko et al., 1999; Ikegami et al., 1998). Intriguingly, the DNA-binding domain in XPA shows structural resemblance to DNA binding β-hairpins (particularly BHD2) in XPC/Rad4p (Min & Pavletich, 2007), which suggests their evolutionary and functional relationship.

So far, the precise roles for the (rather weak) damage-specific DNA-binding activity of XPA remain unclear. XPA reportedly exhibits remarkable binding affinities for DNA containing highly kinked conformations, such as 3-way junctions and the Holliday junction-like structure (Camenisch et al., 2006; Missura et al., 2001). From mutational analyses, it has been

DNA Damage Recognition for Mammalian Global Genome Nucleotide Excision Repair 467

These factors sample all different structural aspects of DNA damage. XPC senses the presence of oscillating unpaired bases, which allows GG-NER to target an extremely broad spectrum of DNA insults. UV-DDB seems more customized for the detection and repair of UV-induced photolesions through direct interaction with the affected bases. As for CPDs (which are refractory to detection by XPC), UV-DDB further extends the substrate specificity of GG-NER. The XPD helicase in TFIIH scans DNA strands as a fine sensor of chemical changes in DNA structure. By integrating these different strategies, GG-NER as a whole can work as a highly versatile, efficient, and accurate system. Numerous biochemical and cell biological studies have confirmed that checks for different structural abnormalities in DNA are conducted in a sequential manner. Additionally, possible stochastic mathematical models have been also discussed (Kesseler et al., 2007; Luijsterburg et al., 2010; Politi et al., 2005). Considering the in vivo situations, decondensation and some remodeling of the chromatin structure would also be expected to precede damage recognition by UV-DDB and XPC, although the underlying mechanism involved in this process remains unclear. These

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a Health and Labour Sciences Research Grant (Research on Intractable Diseases) from the Ministry of Health, Labour and Welfare of

Aboussekhra, A., Biggerstaff, M., Shivji, M.K., Vilpo, J.A., Moncollin, V., Podust, V.N.,

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architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. *Nature,*

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key issues need to be addressed at the molecular level in the near future.

**5. Acknowledgement** 

**6. References** 

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Japan, and by the Takeda Science Foundation.

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proposed that XPA may be suitable for sensing abnormal electrostatic potentials of DNA, which could be caused by certain distorted DNA conformations in the damage-containing DNA duplex that are unwound by the helicase activities of TFIIH (Camenisch et al., 2007). In addition to such "proofreading" functions, our recent DNA-binding assays have raised the possibility that XPA may be required for launching the DNA scanning complex from the XPC-bound sites (Sugasawa et al., 2009). We also have shown that XPA may stimulate the TFIIH helicase activity under certain conditions, presumably through their reported physical interaction (Li et al., 1998; Park et al., 1995). Based on these findings, it is conceivable that a ternary complex involving XPC, XPA, and TFIIH scans DNA strands to search for damage: this model is reminiscent of the damage-recognition mechanism in the bacterial NER system. As for *E. coli*, 2 damage recognition pathways have been proposed (Van Houten et al., 2005): the UvrA homodimer directly recognizes and binds to distorted sites and then recruits UvrB, or preassembled complexes involving 2 UvrA and 1 or 2 UvrB molecules bind DNA in a nonspecific manner and then search for damage by scanning the DNA strands. In this analogy, UvrB seems to correspond to TFIIH as the driving subunit with ATPase/helicase activities, whereas UvrA may have evolved into XPC and/or XPA. Although little amino acid sequence homology exists between these bacterial and mammalian counterparts, the fundamental principles underlying NER damage recognition may have been conserved throughout evolution.

#### **3.4 Implications in the damage surveillance mechanism**

Although the specific DNA binding of UV-DDB and XPC has been observed in vitro, it still remains to be understood how these factors survey DNA and eventually reach relevant sites. For many DNA-binding proteins with sequence- and/or structure-specificity, it has been supposed that the proteins first bind DNA in a nonspecific manner and then "slide" or "hop" to search for their target sites (Gorman & Greene, 2008). A recent report has suggested that BHD1 and BHD2 in XPC may serve as dynamic damage sensors by binding to DNA and rapidly scanning for the integrity of base pairing (Camenisch et al., 2009). Once it encounters a distorted site, BHD3 may be inserted into the duplex to form a stabilized damage-recognition complex.

Apart from these models, our findings that the NER protein complex driven by the XPD helicase can scan DNA strands provides interesting insights into the molecular mechanism underlying in vivo damage surveillance: for instance, the association of XPC even with inappropriate (damage-free) sites could help the NER machinery to survey the local genomic region and find damage at rather distal positions. Possible candidates for such XPC anchoring sites include base mismatches (caused by errors of replication/repair and deamination of bases), thermodynamic "breathing" of the DNA duplex, and other sequences that are intrinsically prone to melting (e.g., transcriptional promoters and replication origins), especially in the presence of topological stresses imposed by chromatin structure. In addition, some endogenous DNA damage, such as abasic sites and single-strand breaks, also may target XPC and thereby launch the "patrolling" system. It would be of great interest to examine how the timing and efficiency of GG-NER are regulated at different genomic loci.

#### **4. Conclusion**

Multiple protein factors are involved in the detection and verification of DNA damage, which, in conjunction with the GG-NER system, determine whether to incise DNA or not. These factors sample all different structural aspects of DNA damage. XPC senses the presence of oscillating unpaired bases, which allows GG-NER to target an extremely broad spectrum of DNA insults. UV-DDB seems more customized for the detection and repair of UV-induced photolesions through direct interaction with the affected bases. As for CPDs (which are refractory to detection by XPC), UV-DDB further extends the substrate specificity of GG-NER. The XPD helicase in TFIIH scans DNA strands as a fine sensor of chemical changes in DNA structure. By integrating these different strategies, GG-NER as a whole can work as a highly versatile, efficient, and accurate system. Numerous biochemical and cell biological studies have confirmed that checks for different structural abnormalities in DNA are conducted in a sequential manner. Additionally, possible stochastic mathematical models have been also discussed (Kesseler et al., 2007; Luijsterburg et al., 2010; Politi et al., 2005). Considering the in vivo situations, decondensation and some remodeling of the chromatin structure would also be expected to precede damage recognition by UV-DDB and XPC, although the underlying mechanism involved in this process remains unclear. These key issues need to be addressed at the molecular level in the near future.

### **5. Acknowledgement**

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by a Health and Labour Sciences Research Grant (Research on Intractable Diseases) from the Ministry of Health, Labour and Welfare of Japan, and by the Takeda Science Foundation.

#### **6. References**

466 DNA Repair

proposed that XPA may be suitable for sensing abnormal electrostatic potentials of DNA, which could be caused by certain distorted DNA conformations in the damage-containing DNA duplex that are unwound by the helicase activities of TFIIH (Camenisch et al., 2007). In addition to such "proofreading" functions, our recent DNA-binding assays have raised the possibility that XPA may be required for launching the DNA scanning complex from the XPC-bound sites (Sugasawa et al., 2009). We also have shown that XPA may stimulate the TFIIH helicase activity under certain conditions, presumably through their reported physical interaction (Li et al., 1998; Park et al., 1995). Based on these findings, it is conceivable that a ternary complex involving XPC, XPA, and TFIIH scans DNA strands to search for damage: this model is reminiscent of the damage-recognition mechanism in the bacterial NER system. As for *E. coli*, 2 damage recognition pathways have been proposed (Van Houten et al., 2005): the UvrA homodimer directly recognizes and binds to distorted sites and then recruits UvrB, or preassembled complexes involving 2 UvrA and 1 or 2 UvrB molecules bind DNA in a nonspecific manner and then search for damage by scanning the DNA strands. In this analogy, UvrB seems to correspond to TFIIH as the driving subunit with ATPase/helicase activities, whereas UvrA may have evolved into XPC and/or XPA. Although little amino acid sequence homology exists between these bacterial and mammalian counterparts, the fundamental principles underlying NER damage recognition

Although the specific DNA binding of UV-DDB and XPC has been observed in vitro, it still remains to be understood how these factors survey DNA and eventually reach relevant sites. For many DNA-binding proteins with sequence- and/or structure-specificity, it has been supposed that the proteins first bind DNA in a nonspecific manner and then "slide" or "hop" to search for their target sites (Gorman & Greene, 2008). A recent report has suggested that BHD1 and BHD2 in XPC may serve as dynamic damage sensors by binding to DNA and rapidly scanning for the integrity of base pairing (Camenisch et al., 2009). Once it encounters a distorted site, BHD3 may be inserted into the duplex to form a stabilized

Apart from these models, our findings that the NER protein complex driven by the XPD helicase can scan DNA strands provides interesting insights into the molecular mechanism underlying in vivo damage surveillance: for instance, the association of XPC even with inappropriate (damage-free) sites could help the NER machinery to survey the local genomic region and find damage at rather distal positions. Possible candidates for such XPC anchoring sites include base mismatches (caused by errors of replication/repair and deamination of bases), thermodynamic "breathing" of the DNA duplex, and other sequences that are intrinsically prone to melting (e.g., transcriptional promoters and replication origins), especially in the presence of topological stresses imposed by chromatin structure. In addition, some endogenous DNA damage, such as abasic sites and single-strand breaks, also may target XPC and thereby launch the "patrolling" system. It would be of great interest to examine how

Multiple protein factors are involved in the detection and verification of DNA damage, which, in conjunction with the GG-NER system, determine whether to incise DNA or not.

the timing and efficiency of GG-NER are regulated at different genomic loci.

may have been conserved throughout evolution.

damage-recognition complex.

**4. Conclusion** 

**3.4 Implications in the damage surveillance mechanism** 


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**24** 

*1Japan*  <sup>2</sup>*USA*

**DNA Double-Strand Break Repair** 

Radhika Pankaj Kamdar1,2 and Yoshihisa Matsumoto1

*2Department of Human Genetics, Emory University, Atlanta, Georgia* 

**Through Non-Homologous End-Joining:** 

**Recruitment and Assembly of the Players** 

*1Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo* 

DNA, this vitally important genetic macromolecule, is under constant assault via endogenous and exogenous agents which cause damage to DNA and thus to cells leading to genomic instability. The primary endogenous cause of DNA damage is caused during continuous replication of DNA at the S phase of the cell cycle effecting spontaneous mutations. Other endogenous DNA damaging agents are reactive oxygen species (ROS) produced as metabolic byproducts. Additionally, breaks are introduced to DNA in the process of recombination, *e.g.*, V(D)J recombination in immune systems and meiotic recombination in reproductive organs. The exogenous DNA damaging agents are ionizing radiations and chemical compounds, which are intercalated into major or minor grooves of

DNA damages include base elimination, modification, cross-linking and strand break. Strand break includes single-strand break (SSB) and double-strand break (DSB). Among these various types of DNA damages, DSB is considered most fatal. Hence healing DSB is vital to circumvent genomic instability encompassing chromosomal aberrations, translocations and tumorigenesis. Eukaryotes have evolved two major pathways to repair DSBs, *i.e.*, homologous recombination (HR) and non-homologous end-joining (NHEJ). This chapter will review the mechanisms of the latter, especially how the players are recruited to

the sites of DSBs and are assembled into multi-protein repair machinery.

**2.1 Homologous Recombination and Non-Homologous End-Joining** 

**2. DNA double-strand break repair through non-homologous end-joining** 

HR is a reaction wherein the genetic material is exchanged between two similar or identical strands of DNA. In the repair of DSB through HR, undamaged DNA serves as a template to reconsitute the original sequence across the break. On the other hand, NHEJ is the direct rejoining of the broken DNA ends without much regard for homology at these ends.

**1. Introduction** 

**pathway** 

DNA strand or form chemical bond with bases.

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