Structure and dynamics of the chromatin remodeler ALC1 bound to a PARylated nucleosome.

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.

Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD.

Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused 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.

Mechanistic Insights into Regulation of the ALC1 Remodeler by the Nucleosome Acidic Patch.

Upon DNA damage, the ALC1/CHD1L nucleosome remodeling enzyme (remodeler) is activated by binding to poly(ADP-ribose). How activated ALC1 recognizes the nucleosome, as well as how this recognition is coupled to remodeling, is unknown. Here, we show that remodeling by ALC1 requires a wild-type acidic patch on the entry side of the nucleosome. The cryo-electron microscopy structure of a nucleosome-ALC1 linker complex reveals a regulatory linker segment that binds to the acidic patch. Mutations within this interface alter the dynamics of ALC1 recruitment to DNA damage and impede the ATPase and remodeling activities of ALC1. Full activation requires acidic patch-linker segment interactions that tether the remodeler to the nucleosome and couple ATP hydrolysis to nucleosome mobilization. Upon DNA damage, such a requirement may be used to modulate ALC1 activity via changes in the nucleosome acidic patches.

DNA surface exploration and operator bypassing during target search.

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 µs. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude.

New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites.

Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC1.

Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.