In contrast, knockout of SMUG1 prevented ALC1 recruitment to chromatin in response to formyl-dU (Figures 6P and 6Q)

In contrast, knockout of SMUG1 prevented ALC1 recruitment to chromatin in response to formyl-dU (Figures 6P and 6Q). Finally, and cells both showed synthetic lethality with loss of BRCA1/2, with a very modest additive effect in double knockouts (Figures 6R and 6S). ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base Pyridoclax (MR-29072) excision by DNA glycosylases but prior to handover to APEX1. Using Pyridoclax (MR-29072) CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused Rabbit Polyclonal to Caspase 6 by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers. and (Bryant et?al., 2005; Farmer et?al., 2005). Despite the notable success of PARPi in the clinic, approximately half of HRD cancers fail to respond to treatment due to innate PARPi resistance, and of those that do respond, 90% ultimately develop acquired PARPi resistance (Noordermeer and van Attikum, 2019). As such, there is an urgent clinical need to identify new therapeutic strategies to improve existing treatments to target HRD and exploit other DNA repair vulnerabilities that exist in cancer, including deficiencies in nonhomologous end joining, mismatch repair, base excision repair (BER), and ATM signaling (Gourley et?al., 2019). DNA of eukaryotic cells is usually compacted into chromatin, and this higher-order complex structure ensures the maintenance of cellular identity. As nucleosomes are perceived as barriers for DNA-related Pyridoclax (MR-29072) processes, they must first be disassembled or re-organized to allow any DNA-templated machinery to access its substrate. While most bulk chromatin packaging occurs during DNA replication, where histones are evicted ahead of the fork and, together with newly synthesized histones, are re-assembled behind the fork (Hammond et?al., 2017), chromatin re-organization outside of S phase impacts on fundamental processes such as transcription and DNA repair. In particular, dynamic changes in chromatin organization occur on damaged chromatin to facilitate timely access of DNA repair enzymes (Price and DAndrea, 2013). Nucleosome eviction and/or sliding are necessary for chromatin relaxation, as well as prompt nucleosome deposition after removal of the DNA lesion (Ransom et?al., 2010). Diverse types of chromatin-remodeling complexes catalyze such chromatin transactions with related ATPase motor translocase domains. Specialized chromatin remodelers involve four subfamilies: imitation switch (ISWI), chromodomain helicase DNA-binding (CHD), switch/sucrose non-fermentable (SWI/SNF), and INO80 (Clapier et?al., 2017; Stadler and Richly, 2017). Different subfamilies preferentially achieve particular outcomes, such as facilitating chromatin access for Pyridoclax (MR-29072) DNA repair transactions, and are targeted to specific chromatin domains via regulatory cues. ALC1 (amplified in liver cancer 1), also known as CHD1L (chromodomain-helicase-DNA-binding protein 1-like), is an ISWI-related chromatin remodeler encoded by a gene on chromosome 1q21, a region commonly amplified in many cancers (Flaus et?al., 2006). ALC1 is differentiated from other members of the ISWI-related remodelers by virtue of a C-terminal macro domain, which possesses high intrinsic affinity for poly(ADP)-ribose (PAR) chains (Ahel et?al., 2009). Through its macro domain, ALC1 is rapidly recruited to sites of DNA damage by PAR chains synthesized by PARP1/2 (Ahel et?al., 2009; Satoh and Lindahl, 1992). Macro domain binding to PAR chains also relieves an autoinhibitory interaction between the macro and ATPase domains of ALC1, which activates ATP hydrolysis and nucleosome sliding (Lehmann et?al., 2017; Singh et?al., 2017). ALC1-dependent chromatin remodeling has been proposed to facilitate DNA repair, but evidence in support of this role is currently lacking (Tsuda et?al., 2017). Moreover, the precise DNA repair and organismal functions of ALC1 remain unknown. Here we show that nucleosome remodeling by ALC1 is required downstream of base excision by DNA glycosylases but upstream of APEX1. Loss of ALC1 leads to toxic BER intermediates that result in single-strand gap formation and replication fork collapse. Since blocking this process in cells confers PARPi sensitization and a critical dependence on HR, our study establishes ALC1 as a potential therapeutic target for treating HRD cancers. Results To investigate the role of ALC1 knockout mouse model from Pyridoclax (MR-29072) a gene trap.

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