Biology subjects

Deindl, S.

Publications and source records attributed to Deindl, S..

5 recordsLinked to original sources

bioRxiv2025

ALC1 Finds a New Foothold on the Nucleosome's Super-Groove

Nucleosomes act as recognition platforms for chromatin-binding factors that coordinate genome maintenance. A distinct structural feature of the nucleosome is the alignment of the DNA major and minor grooves across both gyres, forming continuous major and minor super-grooves. Despite this prominent feature, only one example of a naturally occurring protein binding to a super-groove is documented at the structural level to date. The chromatin remodeler Amplified in Liver Cancer 1 (ALC1) is a key component of the DNA damage response and a promising therapeutic target in cancer. Through extensive classification of our cryo-electron microscopy data, we identified a previously unresolved ALC1-nucleosome complex characterized by a loosely bound conformation of ALC1. This conformation represents an intermediate between the auto-inhibited and active states, and provides new structural insights into the conformational transitions that regulate ALC1 activity. In this intermediate state, the linker of ALC1 engages the nucleosomes minor DNA super-groove across both gyres in a recognition mode observed for the first time in a naturally occurring protein. SynopsisRe-analysis of a publicly available cryo-EM dataset identifies an intermediate state of the oncogenic chromatin remodeler ALC1, revealing the conformational change leading to its activation upon binding to a PARylated nucleosome. The remodeler recognizes the nucleosomes super-groove.

biophysics↗

bioRxiv2021

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 reveal that ALC1 preferentially slides nucleosomes away from DNA breaks.

biophysics↗

bioRxiv2021

Sequence specificity in DNA binding is determined by association rather than dissociation

Sequence-specific binding of proteins to DNA is essential for accessing genetic information. Here, we derive a simple equation for target-site recognition, which uncovers a previously unrecognized coupling between the macroscopic association and dissociation rates of the searching protein. Importantly, this relationship makes it possible to recover the relevant microscopic rates from experimentally determined macroscopic ones. We directly test the equation by observing the binding and unbinding of individual lac repressor (LacI) molecules during target search. We find that LacI dissociates from different target sequences with essentially identical microscopic dissociation rates. Instead, sequence specificity is determined by the efficiency with which the protein recognizes different targets, effectively reducing its risk of being retained on a non-target sequence. Our theoretical framework also accounts for the coupling between off-target binding and unbinding of the catalytically inactive Cas9 (dCas9), showing that the binding pathway can be obtained from macroscopic data. One Sentence SummaryAssociation and dissociation rates are anti-correlated for reactions that include a nonspecific probing step.

biophysics↗

bioRxiv2020

Mechanism of DNA surface exploration and operator bypassing during target search

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 s. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

biophysics↗

bioRxiv2018

Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA1-5. It is however not known how sliding proteins are oriented with respect to DNA in order to recognize specific sequences. Here we measure the polarization of fluorescence emission from single fluorescently labeled lac repressor (LacI) molecules sliding on stretched DNA. Real-time feedback-coupled confocal single-particle tracking allows us to measure fluorescence correlation of the sliding molecules. We find that the fluctuations in the fluorescence signal on the s timescale are accurately described by rotation-coupled sliding on DNA. On average, LacI moves [~]50 base pairs per revolution, which is significantly longer than the 10.5 bp helical periodicity of DNA. Our data support a facilitated diffusion model1 where the transcription factor (TF) scans the DNA grooves for hydrogen bonding opportunities in a pre-aligned orientation with occasional slippage out of the groove.

biophysics↗