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PhD Dissertation Defense - Luay Joudeh

Start Date: April 11, 2017
End Date: April 11, 2017

ADVISOR: Professor Samir Hamdan
TITLE: Toward the reconstitution of the maturation of okazaki fragments multiprotein complex in human at the single molecule level
DATE: Tuesday, April 11, 2017
TIME: 2:00 - 3:00 p.m.
LOCATION: Building 2 · Level 5 · Room 5209

The maturation of Okazaki fragments on the lagging strand in eukaryotes is mediated by a highly coordinated multistep process involving several proteins that ensure the accurate and efficient replication of genomic DNA. Human proliferating cell nuclear antigen (PCNA) that slides on double-stranded DNA is the key player that coordinates the access of various proteins to the different intermediary steps in this process. In this study, I am focusing on characterizing how PCNA recruits and stimulates the structure specific flap endonuclease 1 (FEN1) to process the aberrant double flap (DF) structures that are produced during maturation of Okazaki fragments. FEN1 distorts the DF structures into a bent conformer to place the scissile phosphate into the active site for cleavage. The product is a nick substrate that can be sealed by DNA ligase I whose recruitment is also mediated by its interaction with PCNA. Using single-molecule Förster resonance energy transfer (smFRET) measurements that simultaneously monitored bending and cleavage of various DF substrates by FEN1 alone or in the presence of PCNA, we found that FEN1 and PCNA bends cognate and non-cognate substrates but display remarkable selectivity to stabilize the bent conformer in cognate substrate while promoting the dissociation of non-cognate substrates. This mechanism provides efficiency and accuracy for FEN1 and PCNA to cleave the correct substrate while avoiding the deleterious cleavage of incorrect substrates. This work provides a true molecular level understanding of the key step during the maturation of Okazaki fragment and contributes towards the reconstitution of its entire activity at the single molecule level.
Luay obtained his bachelor degree in Biotechnology with excellence from the Hashemite University of Jordan. Before joining KAUST, he fulfilled a bioinformatics internship at the Sapienza University of Rome. In 2009, He accepted the King’s discovery scholarship to join KAUST as a founding class student to pursue his master degree in biosciences. In spring 2011, he joined Prof Hamdan’s lab, where he played key role in purifying the human protein involved in lagging strand synthesis. He is particularly focusing on utilizing these proteins to systematically reconstitute at the single molecule level the activities involved in linking Okazaki fragments on the lagging strand.