**SiDNA and Other Tools for the Indirect Induction of DNA Damage Responses**

Maria Quanz1,2, Amélie Croset1,2 and Marie Dutreix1 *1Institut Curie, Centre National de Recherche Scientifique (CNRS) UMR3347, Institut National de la Santé et de Recherche Médicale (INSERM) U1021, Université Paris-Sud 11, Centre Universitaire, 91405 Orsay 2DNA Therapeutics SA, 91058 Evry* 

*France* 

### **1. Introduction**

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 type of damage.

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 damage and induce a pure damage-specific response.

alone have been identified in DNA (Cadet et al., 1997) – has led to the evolution of many

SiDNA and Other Tools for the Indirect Induction of DNA Damage Responses 335

The complete signaling network for each damage type and its individual contribution to the cellular damage response are not fully understood, but the essential repair mechanisms have been elucidated (reviewed for example by Fortini & Dogliotti (2007); Friedberg (1995; 2001); Helleday et al. (2008); Li (2008); Wyman & Kanaar (2006)). Figure 1 summarizes the main pathways and highlights the sensors (DNA binding proteins that recognize specific DNA lesions) and transducers (enzymes that amplify the damage signal by posttranslational modification of downstream targets) involved in repair and signaling for particular types of damage. The main DNA damage transducers are the phosphoinositide 3-kinase-like kinase (PIKK) family members ATM (ataxia telangiectasia mutated), ATR (ataxia telangiectasia and Rad3-related) and DNA-PK (DNA-dependent protein kinase). A DNA break signal can also be transduced by poly(ADP-ribose) polymerases 1 or 2 (here both designated PARP), which use NAD<sup>+</sup> to catalyze the modification of their targets with negatively charged, long and branched ADP-ribose polymers. We provide below a brief description of the DNA repair

different repair pathways for sensing and repairing the various types of damage.

pathways, the subsets of damage they repair and the transducers that are activated.

The direct repair of certain alkylation adducts and other uncomplicated base modifications by specialized single enzymes is probably the simplest repair mechanism. O6-alkylguanine DNA alkyltransferase (AGT) is a major enzyme involved in direct repair. It is encoded by the O6-methylguanine-DNA-methyltransferase (MGMT) gene and transfers the alkyl adducts produced by alkylating agents, such as temozolomide, dacarbazine or nitrosourea compounds, from O6-methylguanine, O4-methylthymine, O6-ethylguanine or O6-chloroethylguanine to a cysteine residue within the active site of the enzyme, thereby inactivating the enzyme (Gerson, 2004). Other direct repair enzymes include the DNA dioxygenases ABH2 and ABH3, which can convert 1-methyladenine and 3-methylcytosine back into adenine and cytosine, respectively (Duncan et al., 2002). The repair of alkylated lesions is a rapid process, with most alkylated sites successfully repaired within an hour (Zhu et al., 2009). The types of damage targeted by direct repair processes do not seem to be associated with the activation of damage signaling kinases, probably due to the rapid repair kinetics and the absence of intermediate strand break generation during the repair process.

The base excision repair (BER) pathway recognizes and removes bases carrying non-bulky modifications that have been damaged by nonenzymatic alkylation, oxidation, ring saturation, or IR (Chan et al., 2006). BER also eliminates deaminated bases and DNA single-strand breaks (SSBs). As a first step in BER, a damage-specific DNA glycosylase (e.g. hOOG1, NEIL1 or NEIL2) recognizes and excises the damaged base, leading to the formation of a potentially cytotoxic intermediate apurinic or apyrimidinic site (AP site) (Bandaru et al., 2002; Boiteux & Radicella, 2000). The abasic sugar is cleaved by an AP endonuclease (APE1), which generates a strand break that is further processed by PARP, DNA polymerase *β* and ligase III in either short-patch or long-patch pathways (Fortini & Dogliotti, 2007). PARP not only recognizes the intermediate SSB but also acts as a damage transducer amplifying the damage signal by linking poly(ADP-ribose) (PAR) chains to its substrates, including itself. These polymers bind specific proteins, including XRCC1, DNA ligase III, p53 and DNA-PK,

**2.1 Repair processes that do not directly activate transducers**

**2.2 Repair mechanisms that activate mainly PARP as a transducer**
