Capturing Molecular Movies of DNA Replication and Repair

THUWAL, Kingdom of Saudi Arabia, June 11, 2013 /PRNewswire/ --

King Abdullah University of Science and Technology (KAUST) Assistant Professor of Chemistry and Biology and Principal Investigator of the Laboratory of DNA Replication and Recombination, Dr. Samir M. Hamdan, and his team, have published a groundbreaking paper on the mechanism of flap endonuclease 1 (FEN1), in Cell Reports journal. The other authors of the paper, entitled "Sequential and Multistep Substrate Interrogation Provides the Scaffold for Specificity in Human Flap Endonuclease 1," are: Mohamed A. Sobhy, Luay I. Joudeh, Xiaojuan Huang, and Masateru Takahashi.

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5' nucleases, the superfamily to which FEN1 belongs,represent structure-specific nucleases essential for DNA replication, repair, and recombination. Deficiencies in their genes have been linked to several types of cellular stress and genomic instability.Their outstanding puzzle is that they are highly conserved proteins, yet they recognize a diverse range of RNA and DNA structures and cleave them primarily at the same position relative to a 5' end of a junction.

Structural studies propose a solution for this geometrical puzzle by capturing a DNA bending intermediary step that position the 5' end in the enzyme active pocket and unify the 5' nucleases cleavage site. However, these structures remain a static image of the DNA bending intermediary step based on which speculation was made to address the most important questions regarding how this intermediary step is induced and how 5' nucleases utilize the same intermediary step to recognize diverse range of substrates.

The team from KAUST's Division of Biological and Environmental Sciences and Engineering employed a sophisticated single molecule imaging technique, Frster resonance energy transfer (smFRET), to capture "molecular movies" detailing the structure, dynamics, and reaction mechanisms occurring during this process. The KAUST scientists were able to build a timeline of up to seven intermediary steps before FEN1 commits to catalysis. Such information cannot be accessed through conventional approaches.

These findings will influence how researchers think about the mechanism of other members of 5' nucleases and provide a new concept as to how biological macromolecules can diversify their substrate specificity while maintaining a high degree of structural similarities.

Dr. Hamdan explained that the surprising finding is that FEN1 utilizes a highly complex mechanism that sequentially verifies all substrate features before inducing the superfamily unifying DNA bending intermediary. This sequential and multistep substrate recognition process provides a scaffold that allows different 5' nucleases to recognize different substrates and restrict the induction of DNA bending to the last common step. "We hope that our findings will serve as a base to design inhibitors against FEN1, whose expression is highly correlated with tumor aggressiveness," said Prof. Hamdan.

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Capturing Molecular Movies of DNA Replication and Repair