**2.3.9 XRCC1 interactions with base excision repair DNA intermediates**

XRCC1 is known to play a crucial role in the coordination of two overlapping repair pathways, SSBR and BER (Caldecott, 2003). Although the main role of XRCC1 during BER has been attributed to its participation in the post-incision steps (Wong et al., 2005), which are shared with SSBR, the physical and functional interactions with proteins involved in the initiation of modified bases or abasic sites repair (Marsin et al., 2003, Campalans et al., 2005, Vidal et al., 2001, Wiederhold et al., 2004) suggest that XRCC1 presence at the early steps of BER could be important for assuring a correct repair process. One possible role of XRCC1 could be to optimize the passage of DNA substrates from one enzyme to the next one in the pathway by holding the proteins together through its different interacting domains (Caldecott, 2003).

Investigating the interactions of with different BER DNA intermediates generated either by DNA glycosylase hOGG1 or AP endonuclease APE1 we have found that XRCC1 is able to interact with AP sites via formation of the Schiff base intermediate (Nazarkina et al., 2007). Because hOGG1 possesses both, DNA glycosylase and AP lyase activities, either of two DNA intermediates can be produced: DNA duplex containing an intact AP site or a nick with a 3′ PUA moiety (See Fig. 1). By competition experiments using the THF-containing or regular DNA duplex it was demonstrated that XRCC1 binds DNA with an AP site, or its synthetic analogue, with considerably higher affinity than regular DNA duplex.

XRCC1 is known to bind DNA with single-strand breaks with higher affinity that regular DNA duplex (Mani et al., 2004). We then investigated the relative affinities of XRCC1 to different DNA structures that could be BER intermediates in the repair of single or double DNA lesions. The efficiency of cross-linking is maximal with an incised AP site 3' PUA (Fig. 9A, lane 3). Interestingly, the presence of a strand interruption on the complementary oligonucleotide strongly stimulated the cross-linking of XRCC1 to the AP site. These results suggest that XRCC1 could be important to hold the DNA together during the repair of clustered DNA damage. XRCC1 is also able to cross-link to a 5' dRP residue downstream of a nick (Fig. 9A, lane 6). Comparative analysis of the patterns of protein cross-linking to AP DNA in cell extracts deficient (EM9) and proficient in XRCC1 (EM9-X) (Fig. 9B, lanes 3–6) revealed the product that can be related with XRCC1 (lane 6). Interestingly, that the band is observed with DNA containing interruption opposite AP site (Fig. 9B, lanes 4 and 6).

New Players in Recognition of Intact and Cleaved AP Sites:

radiation.

Biology".

**5. References** 

**4. Acknowledgments** 

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Implication in DNA Repair in Mammalian Cells 325

PARP1 and XRCC1 can regulate initial stage of BER persisting at AP sites until APE1 could come and initiate AP sites cleavage. The PARP1 activation upon interaction with AP sites and resultant automodification appear to facilitate the BER factor recruitment to stimulate the repair process. In the absence or deficiency of APE1, PARP1 can provide temporal protection of AP sites. Ku antigen can temporarily protect AP sites situated within 1-2 turns of DNA helix from double-strand breaks or displays AP/dRP lyase activity near doublestrand ends. The last activity has no analogues and is indispensable for proper repair of DS breaks with particular blocking groups at the ends, which can result from action of ionizing

In other case of revealed AP/dRP lyase activities, they appear to function under particular physiologic or stressful conditions modulating the capacity of the BER system or providing the appropriate functions of the repair machinery when some participants are missed or inactive. Interestingly, that structural chromatin proteins –HMGB1 and HMGA – were found to interact with AP sites displaying an enzymatic activity. Although being a relative weak, this activity may be biologically significant due to high abundance of these proteins.

This work was supported in part by the Russian Foundation for Basic Research (Project No 10-04-01083) and Program of the Russian Academy of Sciences "Molecular and Cellular

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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 the asterisk.

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 EM9 one (lane 7).

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 with XRCC1 binding to AP DNA.

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 until the next enzyme recruited by XRCC1 is able to process it.
