03 Feb Humanized Fly Model Allows Deeper Insights Into Rare Genetic Syndrome

MedicalResearch.com Interview with:

Dr. Chesnokov

Igor Chesnokov, Ph.D
Department of Biochemistry and Molecular Genetics
School of Medicine
University of Alabama at Birmingham, Alabama

MedicalResearch.com: What is the background for this study?

Response: DNA replication is fundamentally important for tissue development, growth and homeostasis. Impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Meier-Gorlin Syndrome (MGS) is an autosomal recessive disorder that is also known as ear, patella, short stature syndrome and/or microtia, absent patella, micrognathia syndrome, traits highlighting the core clinical phenotypes.

The genes affected by MGS mutations include many members of pre-replicative complex (pre-RC), such as Origin Recognition Complex (ORC) subunits (Orc1, Orc4, Orc6), Cdc6, Cdt1, CDC45, MCM5 as well as Geminin, suggesting that the clinical phenotype is caused by defects in DNA replication initiation. As the pre-RC complex is essential for DNA replication, the mutations in its components are expected to impair cell proliferation and reduce growth.

The smallest subunit of ORC, Orc6, is the most divergent and enigmatic among ORC subunits. Orc6 is important for DNA replication in all species. Metazoan Orc6 proteins consist of two functional domains: a larger N-terminal domain important for binding of DNA and a smaller C-terminal domain important for protein-protein interactions. A mutation coding for a tyrosine 232 to serine alteration (Y232S) in the C-terminal domain of Orc6 is linked to MGS in humans. Recently, a new Orc6 mutation was described that also resulted in MGS. Unlike the previously described MGS mutation, this amino acid substitution, Lysine 23 to Glutamic acid (K23E), localizes in the N-terminal domain of Orc6. 

MedicalResearch.com: What are the main findings?

Response: These two Orc6-based MGS mutations are interesting to contrast, because position 23 is near the front, or the N-terminal domain, of the long chain of connected amino acids that folds to form the Orc6 protein. Position 232 is near the end, or the C-terminal domain, of the Orc6 protein strand. In our previous research on the Y232S (225 in Drosophila) mutation (2015 article in American Journal of Medical Genetics), we found that the C-terminal domain of Orc6 is important for protein-protein interactions to help build the ORC complex. In the current study, we discovered that the K23E mutation in the N-terminal domain of Orc 6 disrupts the protein’s ability to bind to DNA. This specific binding is a vital step in ORC function, necessary for the formation of the pre-Replication Complex.

Thus, although the two mutations have different underlying molecular mechanisms, they both cause deficient pre-replicative complex formation and reduced DNA replication, and they produce a similar phenotype in MGS patients.

MedicalResearch.com: What should readers take away from your report?

Response: One key in this research was to create a “humanized” model of the Meier-Gorlin Syndrome in Drosophila that allows studying the functions of the human protein in a live animal system. We also created chimeric Orc6 genes to produce a hybrid Orc6 that was human in the N-terminal domain and fruit fly in the C-terminal domain. This chimeric transgene was able to completely rescue the fruit flies with an orc6 null mutant background, and they grew into adults that were undistinguishable from fruit flies with wild-type Orc6. This hybrid Orc6 then was used to test the K23E mutation in fruit flies and study its molecular mechanism. Using these “humanized” fly strains carrying the K23E MGS mutation, we identified a molecular mechanism resulting in a MGS phenotype and compared it with our Y225S MGS fly model. We found that despite having different underlying molecular mechanisms both MGS mutations resulted in similar phenotypes, deficient pre-RC formation and reduced DNA replication.

MedicalResearch.com: What recommendations do you have for future research as a result of this work?

Response: The hybrid approach, described in our study, revealed the importance of evolutionarily conserved and variable domains of the Orc6 protein and allowed the investigation of human protein functions and the analysis of the critical amino acids in a live animal system. We believe that a hybrid approach not only open a broad avenue to study new Orc6 mutations for medical and general science purposes, but the approach might also be useful in other humanized models. In summary, the humanized fly model presented in our studies has the unique advantage of being able to differentially test both fly, human and chimeric Orc6 proteins to reveal conserved and divergent features of the protein and its functions in the cells of metazoan organisms.

The fly strains generated during this work will be useful in analyzing the functions of human protein in a convenient heterologous system. Specifically, our studies revealed novel insights into molecular mechanisms underlying MGS pathology and provide important clues about disease origin and development.

Citation:

Humanized Drosophila Model of the Meier-Gorlin Syndrome Reveals Conserved and Divergent Features of the Orc6 Protein
Maxim Balasov, Katarina Akhmetova and Igor Chesnokov
GENETICS December 1, 2020 vol. 216 no. 4 995-1007; https://doi.org/10.1534/genetics.120.303698 

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Last Updated on February 4, 2021 by Marie Benz MD FAAD