Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome
- Uppsala University, Sweden
- St Jude Children's Research Hospital, United States
- The Francis Crick Institute, United Kingdom
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 reveal that ALC1 preferentially slides nucleosomes away from DNA breaks.
Data availability
The cryo-EM map of the ALC1-nucleosome complex in the active state was deposited at the EMDB with accession code EMD-13065. The model of the ALC1-nucleosome complex in the active state was deposited at the PDB with accession code 7OTQ. The map series from the cryoDRGN graph traversal was deposited at the EMDB with accession code EMD-13070. Raw movies, extracted particles and their coordinates, and cryoDRGN and cryoSPARC job directories were deposited in EMPIAR with accession code EMPIAR-10739.
Article and author information
Author details
Guillaume Gaullier
Funding
European Research Council (714068)
- Sebastian Deindl
Knut och Alice Wallenbergs Stiftelse (KAW 019.0306)
- Sebastian Deindl
Vetenskapsrådet (VR Grant 2019-03534)
- Sebastian Deindl
Cancerfonden (19 0055 Pj)
- Sebastian Deindl
Cancer Research UK (FC0010048)
- Simon Boulton
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Bacic et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome
eLife 10:e71420.
https://doi.org/10.7554/eLife.71420
Further reading
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- Chromosomes and Gene Expression
Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.
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- Chromosomes and Gene Expression
The association between late replication timing and low transcription rates in eukaryotic heterochromatin is well known, yet the specific mechanisms underlying this link remain uncertain. In Saccharomyces cerevisiae, the histone deacetylase Sir2 is required for both transcriptional silencing and late replication at the repetitive ribosomal DNA (rDNA) arrays. We have previously reported that in the absence of SIR2, a de-repressed RNA PolII repositions MCM replicative helicases from their loading site at the ribosomal origin, where they abut well-positioned, high-occupancy nucleosomes, to an adjacent region with lower nucleosome occupancy. By developing a method that can distinguish activation of closely spaced MCM complexes, here we show that the displaced MCMs at rDNA origins have increased firing propensity compared to the nondisplaced MCMs. Furthermore, we found that both activation of the repositioned MCMs and low occupancy of the adjacent nucleosomes critically depend on the chromatin remodeling activity of FUN30. Our study elucidates the mechanism by which Sir2 delays replication timing, and it demonstrates, for the first time, that activation of a specific replication origin in vivo relies on the nucleosome context shaped by a single chromatin remodeler.
