Abstract
The chromatin remodeler ALC1 is recruited to and activated by DNA damage-induced poly(ADP-ribose) (PAR) chains deposited by PARP1/PARP2/HPF1 upon detection of DNA lesions. ALC1 has emerged as a candidate drug target for cancer therapy as its loss confers synthetic lethality in homologous recombination-deficient cells. However, structure-based drug design and molecular analysis of ALC1 have been hindered by the requirement for PARylation and the highly heterogeneous nature of this post-translational modification. Here, we reconstituted an ALC1 and PARylated nucleosome complex modified in vitro using PARP2 and HPF1. This complex was amenable to cryo-EM structure determination without cross-linking, which enabled visualization of several intermediate states of ALC1 from the recognition of the PARylated nucleosome to the tight binding and activation of the remodeler. Functional biochemical assays with PARylated nucleosomes highlight the importance of nucleosomal epitopes for productive remodeling and suggest that ALC1 preferentially slides nucleosomes away from DNA breaks.
Highlights
DNA double-strand breaks (DSBs) are among the most cytotoxic DNA lesions, and a common strategy in cancer therapy is to overwhelm the repair capacity of cancer cells with excess DSBs using radiation or cytotoxic chemotherapies
ALC1 uses its macro domain to bind to PAR (Ahel et al, 2009; Gottschalk et al, 2009) produced by PARP1 and PARP2
While an essentially full-length protein (ALC1fl) did bind to these nucleosomes (Figure 1A, lane 4), this binding predominantly originated from non-specific interactions between its macro domain (ALC1macro) and DNA (Figure 1A, lane 3), in agreement with our previous results (Lehmann et al, 2017)
Summary
DNA double-strand breaks (DSBs) are among the most cytotoxic DNA lesions, and a common strategy in cancer therapy is to overwhelm the repair capacity of cancer cells with excess DSBs using radiation or cytotoxic chemotherapies. As a crucial step in the DNA damage response, activation of ALC1 most likely requires nucleosomal histone PARylation in the presence of HPF1. In the absence of cross-linking, this complex yielded cryo-EM data of sufficient quality to permit structure determination of the active state of the nucleosome-bound remodeler, and to visualize multiple conformational states visited during substrate recognition. Examination of these states identified physical interactions between ALC1 and several nucleosomal epitopes, which our functional assays confirm to be critical for remodeling
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