Biology subjects

Chua, G. N. L.

Publications and source records attributed to Chua, G. N. L..

4 recordsLinked to original sources

bioRxiv2025

Molecular interplay between the DNA damage checkpoint kinase Mec1-Ddc2 and its activator Dpb11 on gapped DNA

The eukaryotic DNA damage and replication stress checkpoint is initiated by activation of the apical kinase complex ATR-ATRIP on RPA-coated ssDNA. In Saccharomyces cerevisiae, the Mec1-Ddc2 (hATR-ATRIP) activator and checkpoint mediator Dpb11 (hTopBP1) is recruited to the 9-1-1 checkpoint clamp (another Mec1-Ddc2 activator) at 5 ss-dsDNA junctions. It remains unclear how Mec1-Ddc2 encounters its activators on damaged DNA due to their differential DNA binding preferences. Using real-time single-molecule imaging, we show that Dpb11 binds to ssDNA directly and localizes to ss-dsDNA junctions in an RPA-dependent manner. Furthermore, Dpb11 recruits Mec1-Ddc2 to ss-dsDNA junctions. Single-molecule force spectroscopy was used to demonstrate that Dpb11 forms bridges on ssDNA, both alone and in the presence of RPA, reducing the end-to-end distance of gapped DNA. These data support a model in which Dpb11 facilitates Mec1-Ddc2 colocalization with its activators directly by recruiting Mec1-Ddc2 to gap junctions and indirectly by decreasing the effective gap length.

biophysics↗

bioRxiv2025

A non-catalytic role for RFC in PCNA-mediated processive DNA synthesis

The ring-shaped sliding clamp PCNA enables DNA polymerases to perform processive DNA synthesis during replication and repair. The loading of PCNA onto DNA is catalyzed by the ATPase clamp loader RFC. Using a single-molecule platform to visualize the dynamic interplay between PCNA and RFC on DNA, we unexpectedly discovered that RFC continues to associate with PCNA after loading, contrary to the conventional view. Functionally, this clamp-loader/clamp complex is required for processive DNA synthesis by polymerase {delta} (Pol{delta}), as the PCNA-Pol{delta} assembly is inherently unstable. This architectural role of RFC is dependent on the BRCT domain of Rfc1, and mutation of its DNA-binding residues causes sensitivity to DNA damage in vivo. We further showed the FEN1 flap endonuclease can also stabilize the PCNA-Pol{delta} interaction and mediate robust synthesis. Overall, our work revealed that, beyond their canonical enzymatic functions, PCNA-binding proteins harbor non-catalytic functions essential for DNA replication and genome maintenance.

biophysics↗

bioRxiv2023

Differential dynamics specify MeCP2 function at methylated DNA and nucleosomes

Methyl-CpG-binding protein 2 (MeCP2) is an essential chromatin-binding protein whose mutations cause Rett syndrome (RTT), a leading cause of monogenic intellectual disabilities in females. Despite its significant biomedical relevance, the mechanism by which MeCP2 navigates the chromatin epigenetic landscape to regulate chromatin structure and gene expression remains unclear. Here, we used correlative single-molecule fluorescence and force microscopy to directly visualize the distribution and dynamics of MeCP2 on a variety of DNA and chromatin substrates. We found that MeCP2 exhibits differential diffusion dynamics when bound to unmethylated and methylated bare DNA. Moreover, we discovered that MeCP2 preferentially binds nucleosomes within the context of chromatinized DNA and stabilizes them from mechanical perturbation. The distinct behaviors of MeCP2 at bare DNA and nucleosomes also specify its ability to recruit TBLR1, a core component of the NCoR1/2 co-repressor complex. We further examined several RTT mutations and found that they disrupt different aspects of the MeCP2-chromatin interaction, rationalizing the heterogeneous nature of the disease. Our work reveals the biophysical basis for MeCP2s methylation-dependent activities and suggests a nucleosome-centric model for its genomic distribution and gene repressive functions. These insights provide a framework for delineating the multifaceted functions of MeCP2 and aid in our understanding of the molecular mechanisms of RTT.

biophysics↗

bioRxiv2021

Single-stranded nucleic acid sensing and coacervation by linker histone H1

The linker histone H1 is the most abundant group of eukaryotic chromatin-binding proteins. The mechanism underlying the diverse physiological functions of H1 remains unclear. Here we used single-molecule fluorescence and force microscopy to observe the behavior of H1 on DNA under different tensions. Unexpectedly, we found that H1 coalesces around nascent ssDNA. Molecular dynamics simulations revealed that multivalent and transient interactions between H1 and ssDNA mediate their phase separation. We further showed that longer and unpaired nucleic acids result in more viscous, gel-like H1 droplets. Finally, we imaged H1 puncta in cells under normal and stressed conditions and observed that RPA and H1 occupy separate nuclear regions. Overall, our results provide a new perspective to understanding the role of H1 in genome organization and maintenance.

biophysics↗