**5. References**

324 Selected Topics in DNA Repair

Fig. 9. Interactions of XRCC1 with BER DNA intermediates (From Nazarkina et al., 2007). (A) Cross-linking of XRCC1 to different 5'-32P-labeled AP DNA. DNA containing intact AP sites (lanes 1, 2, 4, and 5) and cleaved AP site (3' PUA) (lane 3) were 5' end labeled, while DNA containing hydrolyzed AP site (5' dRP) (lane 6) was 3' end labeled. (B) Cross-linking of XRCC1 with AP DNA in CHO cell extracts deficient in XRCC1 (EM9) or EM9 expressing His-tagged human XRCC1 (EM9-9). Position of radioactive label in DNA is designated by

To confirm that this product corresponds to the XRCC1 containing conjugate, after trapping with NaBH4, His-tagged XRCC1 was recovered by pull-down using a Ni-NTA resin. After this purification step, the product was recovered from the EM9-X extract (lane 8) but not the

Taken together, these results demonstrate XRCC1 ability to interact with intact and cleaved AP sites, including cell extracts, e.g. in the presence of cellular proteins that can interfere

Thus, using the Schiff-base-mediated cross-linking, we show that XRCC1 displays a specific affinity for AP containing substrates. Although at this time we cannot evaluate the in vivo relevance of covalent complexes between XRCC1 and DNA, considering the Schiff base reversibility, it is tempting to speculate that its formation during BER of AP sites could be a physiological response to situations where a reactive intermediate needs to be protected

Combination of affinity based cross-linking of proteins with specific DNA probes containing intact or cleaved AP sites and MS analysis allowed to identify new players in recognition/processing of these ubiquitous lesions. Some identified proteins are known as regulatory proteins of specific DNA repair processes, not necessarily involved in repair of AP sites, but all these proteins are related to cell radiosensitivity. Two of them—XRCC1 and PARP1—belong to the base excision repair system, but have been previously considered as participants of later stages of the BER process. By virtue of its interaction with AP sites,

the asterisk.

EM9 one (lane 7).

**3. Conclusion** 

with XRCC1 binding to AP DNA.

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**Part 3** 

**Methods in DNA Repair** 


**Part 3** 

**Methods in DNA Repair** 

330 Selected Topics in DNA Repair

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**1. Introduction**

type of damage.

damage and induce a pure damage-specific response.

Cells respond to DNA damage by activating an intricate signaling network leading to DNA repair, cell cycle arrest or apoptosis. In recent years, progress has been made in the discovery and characterization of a number of DNA repair pathways, and it has become apparent that the inhibition of specific components of these pathways could offer new targets for combating the resistance of tumors to chemotherapy or radiotherapy. A thorough understanding of the various DNA repair pathways and their regulation is therefore essential. The DNA damage response (DDR) is of great importance in determining cell fate decisions. It includes many signal amplification steps and several steps that are partly redundant due to the ability of different kinases to phosphorylate the same target. Furthermore, the timing and origin of the damage play an important role in determining the DNA repair pathway activated. All this makes it difficult to study the role of one particular protein in DNA damage signaling. In addition, the available tools for activating DNA repair pathways are mostly agents that systematically produce more than one type of DNA damage. Even if the damage caused is initially of one predominant type (as for topoisomerase inhibitors, alkylators or the I-SceI endonuclease system), the damage may rapidly be transformed by normal cellular processes, such as DNA replication, or specific nuclease activities. Studies of the DDR become even more complicated if the agent used to create DNA lesions also damages other cellular components, as is the case for ionizing radiation (IR), alkylators and hydrogen peroxide. Furthermore, the damage is transient, as DNA damage signaling is rapid and lesions are quickly repaired. The signal induced by the damage therefore disappears rapidly, soon after the induction of damage. In some cells, the DNA may not be successfully repaired, leading to apoptosis or senescence. These aspects make it difficult to study the signaling network induced by a given

**SiDNA and Other Tools for the Indirect** 

**Induction of DNA Damage Responses** 

*1Institut Curie, Centre National de Recherche Scientifique (CNRS) UMR3347, Institut National de la Santé et de Recherche Médicale (INSERM) U1021,* 

Maria Quanz1,2, Amélie Croset1,2 and Marie Dutreix1

*Université Paris-Sud 11, Centre Universitaire, 91405 Orsay* 

*2DNA Therapeutics SA, 91058 Evry* 

*France* 

**15**

In this chapter, we will provide an overview of the response of the cell to DNA damage and possible ways of inducing a DDR in cells without actually damaging chromatin. We will focus on stabilized short interfering DNA molecules (siDNA), which mimic different types of
