Alfredo De Biasio
Molecular architecture and function of DNA replication and repair machines
The genetic information is encoded in long chains of deoxyribonucleic acid (DNA) molecules packaged into chromosomes in the cell nucleus. Before dividing, cells need to accurately duplicate their DNA, to ensure that each daughter cell has an identical copy. Critical players in DNA replication are the so-called DNA clamps, ring-shaped proteins that slide on the DNA double helix and anchor the polymerases, the enzymes that replicate DNA, to the genomic template. Beside DNA replication, sliding clamps function in the DNA damage response and the maintenance of genome integrity. Due to their central role in cell proliferation, DNA clamps are an important hallmark of tumors, as well as potential drug targets for anti-cancer therapy.
Our aim is to unravel the molecular architecture of eukaryotic DNA replication complexes involving DNA sliding clamps, and to shed light on their inner workings. To tackle these composite, supramolecular assemblies we integrate structural (X-ray, NMR, Cryo-Electron Microscopy), biochemical and computational methods.
In our work, we have determined the structure of the human clamp PCNA bound to p15PAF, a modulator of the DNA damage tolerance pathway, revealing a unique mode of binding with functional implications. Our work has also provided a structural basis for the mechanism of PCNA sliding on DNA.
Our current research exploits the power of single-particle Cryo-EM to unravel the structure and mechanism of the human holoenzymes that replicate normal and damaged DNA. We have recently determined the Cryo-EM structure of the human Okazaki fragment processing complex, which includes the lagging strand DNA polymerase δ, primed DNA, PCNA and flap endonuclease 1 (FEN1). We have also determined the structure of human Pol κ-PCNA-DNA complex, the holoenzyme that replicates DNA through regions of damage.
- Lancey C., Tehseen M., Takahashi M., Sobhy M.A., Ragan T.J., Crehuet R., Hamdan S.M.* and De Biasio A.* “Cryo-EM structure of Pol κ DNA-PCNA holoenzyme and implications for polymerase switching in DNA lesion bypass” Preprint at https://www.biorxiv.org/content/10.1101/2020.07.10.196956v1 *corresponding author
- Lancey C, Tehseen M, Raducanu V-S, Rashid F, Merino N, Ragan TJ, Savva CG, Zaher MS, Shirbini A, Blanco FJ, Hamdan SM, De Biasio A. Structure of the processive human Pol δ holoenzyme. Nature Communications 11, 1109 (2020) *corresponding author ** Paper recommended by F1000Prime
- De March M., Merino N., Barrera-Vilarmau S., Crehuet R., Onesti S.*, Blanco F.J.* and De Biasio A.* (2017) “Structural basis of human PCNA sliding on DNA” Nat. Commun. 7, 13935 ** *corresponding author ** this paper was reviewed in Yao N. and O'Donnell M. “DNA Replication: How Does a Sliding Clamp Slide?” (2017) Curr. Biol. 27(5):R174-R176
- De Biasio A.*, Ibañez de Opakua A., Mortuza G., Molina A., Cordeiro T.N., Castillo F., Villate M., Merino N., Delgado S., Gil-Cartón D., Luque I., Diercks T., Bernadó P., Montoya G. and Blanco F.J.* (2015) “Structure of p15PAF‒PCNA complex and implications for clamp sliding during DNA replication and repair” Nat. Commun. 6:6439 *corresponding author
Taha Shahid, Souvika Bakshi, Claudia Lancey and Kerry Blair