We demonstrate enhanced recruitment of BRCA1 to etoposide-damaged DNA

We demonstrate enhanced recruitment of BRCA1 to etoposide-damaged DNA. al., 2010; Symington and Gautier, 2011). Although DSBs are primarily repaired by nonhomologous end joining (NHEJ), they can also be resolved during S phase by homology-directed repair (HDR) using the sister chromatid as a DNA template (Symington and Gautier, 2011; Chapman et al., 2012; Andres et al., 2014; Aparicio et al., 2014). The decision to use NHEJ or HDR is governed in part by DNA resection, a nucleolytic process in which DSB ends are converted into 3 single-strand DNA CI 972 overhangs, an essential intermediate for the downstream steps of CI 972 HDR and a potent inhibitor of NHEJ. DNA resection in eukaryotes is initiated by CtIP (Sae2 in yeast) and the MRN/X complex (Mre11, Rad50, and Nbs1/Xrs2 in yeast; Sartori et al., 2007; Huertas and Jackson, 2009; Qvist et al., 2011). Whereas MRN-CtIP mediates short-range 5 to 3 resection, exonuclease 1 (Exo1) can, after a lag, extensively resect DSBs independently of MRN-CtIP. The resection activity of CtIP is regulated in both a cell-cycleC and damage-dependent manner that is conserved among vertebrates (You et al., 2009; Peterson et al., 2011). Interestingly, CtIP also binds the BRCA1 tumor suppressor (Yu et al., 1998), an essential HDR protein. Whereas the effect of BRCA1 on CtIP-mediated DNA resection remains unclear (Reczek et al., 2013; Zhou et al., 2014), genetic data suggest a role for CtIPCBRCA1 interaction in cellular tolerance to camptothecin and, to a lesser extent, to etoposide (Nakamura et al., 2010). Topoisomerases facilitate DNA transactions such as replication and transcription by relieving DNA topological stress. Type IB topoisomerases (Top1) remove supercoils by generating single-strand DNA breaks that allow DNA to rotate over its CI 972 axis. Through a CI 972 transesterification reaction, the catalytic tyrosine of the enzyme forms a transient phosphotyrosine covalent linkage, generating a nick in the DNA. After isomerization, the DNA phosphodiester backbone is restored when the 5 OH of the broken DNA strand attacks the 3 phosphotyrosine bond, liberating Top1 for subsequent cleavage and unwinding. Type IIA topoisomerases (Top2) remove topological constraints by generating staggered incisions, 4 bp apart, on both strands of DNA, which allow passage of a second DNA duplex through the DSB (Liu et al., 1983; Rowe et al., 1984; Wu et al., 2011). This reaction also entails formation of a transient proteinCDNA adduct, in this case between a tyrosine residue at each active site of the Top2 dimer and the 5 phosphates of DNA strands on both sides of the DSB. After isomerization, the resulting 3-hydroxyl DNA ends direct the reversal of the phosphotyrosyl bonds, thereby enabling the release of the topoisomerase and religation of the DNA break (Pommier et al., 2010; Vos et al., 2011). Given that DNA breaks are normal intermediates of topoisomerase activity, abortive topoisomerase reactions that stabilize the transient proteinCDNA adduct represent a significant source of DNA damage (Vos et al., 2011). Moreover, the formation of such trapped proteinCDNA adducts can be exacerbated by topoisomerase poisons such as etoposide (also known as VP-16-213), which increases the stability of Top2CDNA adducts (Pommier et al., 2010). These unprocessed Top2CDNA adducts block DNA replication and RNA transcription and generate lethal DSBs that can induce cell-death pathways. Because cancer cells rely more heavily Rabbit Polyclonal to GPR142 on DNA repair than normal cells (Tewey et al., 1984; Treszezamsky et al., 2007; Nitiss, 2009), the cellular toxicity of etoposide has been exploited therapeutically for a variety of human malignancies, including small cell lung carcinoma, testicular cancer, and lymphomas. Eukaryotic cells coordinate multiple pathways to eliminate trapped proteinCDNA adducts. Whereas ubiquitin-dependent proteasome degradation can remove much of the adducted proteins, further enzymatic processing by a DNA repair pathway is required to release the residual adducted peptide. For example, formaldehyde-induced DNACprotein cross-links require nucleotide-excision repair or HDR for resolution (Ide et al., 2011). Similarly, Top2-adducted DNA ends can be converted into ligatable ends upon direct cleavage of the 5-tyrosine.

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