Miller-Browne, V. | bioRxiv Science

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↗

bioRxiv2020

MCOLN1 gene-replacement therapy corrects neurologic dysfunction in the mouse model of mucolipidosis IV.

Mucolipidosis IV (MLIV, OMIM 252650) is an orphan disease leading to debilitating psychomotor deficits and vision loss. It is caused by loss-of-function mutations in the MCOLN1 gene that encodes thethe lysosomal transient receptor potential channel mucolipin 1 (TRPML1). With no existing therapy, the unmet need in this disease is very high. Here we show that AAV-mediated gene transfer of the human MCOLN1 gene rescues motor function and alleviates brain pathology in the Mcoln1-/- MLIV mouse model. Using the AAV-PHP.b vector for initial proof-of-principle experiments in symptomatic mice, we showed long-term reversal of declined motor function and significant delay of paralysis. Next, we designed self-complimentary AAV9 vector for clinical use and showed that its intracerebroventricular administration in post-natal day 1 mice significantly improved motor function and myelination and reduced lysosomal storage load in the MLIV mouse brain. We also showed that CNS targeted gene transfer is necessary to achieve therapeutic efficacy in this disease. Based on our data and general advancements in the gene therapy field, we propose scAAV9-mediated CSF-targeted MCOLN1 gene transfer as a therapeutic strategy in MLIV.

genetics↗