We seek to elucidate the function of the essential DNA replication factor Cdt1. The correct execution of DNA replication is critical for normal cell proliferation. Understanding the molecular mechanisms of how this process is regulated via Cdt1 is the focus of this study. Gaps in the field include understanding precisely how replication factors assemble prereplication complexes (pre-RC) and load DNA helicases at origins of DNA replication. Cdt1 is essential for these events, yet the molecular mechanisms of its activity are still remarkably mysterious. This project focuses on understanding Cdt1 function using naturally occurring disease-associated mutations to shed light on Cdt1 mechanistic function. The mutations used in this study are found in Meier- Gorlin (MG) syndrome patients and in human cancers. MG syndrome is a dwarfism disorder characterized by tissue-specific hypoplasias. Patients are overall small, suffering from cell proliferation defects.
Our first aim takes advantage of mutations found in different locations of the Cdt1 protein potentially perturbing protein- protein interactions. Based on the location along the Cdt1 protein, we hypothesize that Cdt1 requires precise intermolecular interactions with known binding partners for properly regulated pre-RC formation and helicase loading. We are testing this idea using human cell culture systems and biochemical assays to determine the phenotypic consequences of each mutation on cell proliferation and protein-protein interactions. Preliminary results suggest the presence of a novel helicase biding domain on Cdt1, as well as novel regulatory mechanisms that will advance our current knowledge in the field.
Our second aim seeks to fill our knowledge gap linking tissue-specific cell proliferation with whole organism development. Although MG patients have an overall cell proliferation defect, some tissues are more affected than others. We will use D. melanogaster to fill this knowledge gap. Given the phenotypes seen in Meier-Gorlin syndrome and human cancers, we hypothesize that different tissues in the animal will be differentially sensitive to cell proliferation defects during development. We will use the CRISPR/Cas9 system to knock-in Cdt1 mutations in Drosophila. We will then examine the morphological effects of each Cdt1 mutation during embryogenesis, tissue development, and overall health of the organism. Developmental abnormalities we observe will be linked to defects in cell proliferation on the tissue being observed and the properties of the Cdt1 mutation being assayed. Ultimately, this study will advance our understanding of the mechanisms involved in pre-RC formation and helicase loading, which are required for the correct execution of DNA replication and cell proliferation. Additionally, the study has the potential to reveal separation-of-function alleles as well as novel molecular mechanisms regulating Cdt1 function during cell proliferation and tissue-specific development in the context of a whole organism.

Public Health Relevance

Genome instability arising from errors in DNA replication during the cell division cycle is a major contributor to cell proliferation defects seen in developmental disorders and cancers. The human Cdt1 protein is intimately involved in forming the complexes and recruiting the DNA helicases required for the initial steps of DNA replication. Understanding exactly how these processes are regulated can lead to the development of therapeutic interventions to halt or abrogate developmental disorders and cancer progression. The goal of this project is to understand precisely how Cdt1 works during the initial steps of DNA replication. This new knowledge will help us understand not only the biology of Cdt1 but also to devise potential cures and treatments to diseases where problems in DNA replication lead to developmental disorders and cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31GM121073-03
Application #
9599463
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Brown, Patrick
Project Start
2016-12-01
Project End
2019-05-31
Budget Start
2018-12-01
Budget End
2019-05-31
Support Year
3
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599