Researchers from the Hong Kong University of Science and Technology (HKSUST) and the University of Hong Kong (HKU) recently showed that the selective determinant of the Origin Recognition Complex (ORC) for DNA-binding lies in the 19-amino acid insertion helix in the Orc4 subunit found in yeast but absent in human. Removing this model from Orc4 transforms the yeast ORC, which selects origins based on the binding of the base at specific sites, to a site whose selectivity is determined by the chromatin view (genetic nucleus profile), a distinct feature shared by human ORC.
Further understanding of preferred DNA profiles and nuclear positioning requirements will provide new insights into human ORC plasticity in selecting replication initiation sites during programmed development and disease transformation, as well as assist in identifying potential targets for anticancer drug screening and treatment design.
All organisms, from the simple unicellular yeast to the complex multicellular human being, spread through cell divisions. Each section requires the exact replication of the genomic DNA, which is a blueprint for the identity of each organism. Excessive reproduction may lead to cancer and a lack of reproduction may lead to developmental defects such as Meyer-Guerlain syndrome. DNA replication begins at the origins of replication by ORC and other protein complexes assembled at these sites.
Interestingly, ORC is highly conserved in protein structure and function from yeast to human, but it targets DNA sites that do not have clear common features in these two systems. What causes this difference? This question has puzzled scientists for decades.
“The structure teaches function,” said Professor Peak Tay, Principal Investigator and Senior Member at the Institute for Advanced Study, HKUST. “In 2018, we resolved the high-resolution structure of ORC yeast using cryogenic electron microscopy. When we compared the structures between yeast and humans, we found one important difference between them, which lies in one subunit of ORC – Orc4.”
Professor Tai said, “Orc4 yeast contains 19 additional amino acids that make specific contacts with the original DNA and are not found in humans.” “We immediately realized that this short form of Orc4 might be a critical factor causing the different behaviors of ORC in DNA binding between yeast and human. Then we deleted this model in yeast cells to test whether it could transform the yeast ORC into one that behaves with human-like properties. In fact, an engineered ORC in yeast behaves like human ORC, or as we call it, humanized ORC. ”
DNA replication is one of the most fundamental processes in living cells. They are catalyzed by a similar set of yeast-to-human transcription mechanisms. In yeast cells, DNA replication begins from a set of sites, called the origins of replication. These sites share a specific DNA sequence, which can be identified by a protein complex of six subunits known as the Origin Recognition Complex (ORC), Orc1-6. The ORC binds to the original DNA, and then acts as a platform for Mcm2-7 Helicase recruitment into the DNA. The Mcm2-7 helicase is the machine responsible for double-stranded DNA separations to provide templates for DNA replication.
“The activities of each parent are strictly regulated to ensure the integrity of the genome,” said Professor ZHAI Yuanliang, study collaborator and associate professor at the School of Biological Sciences, College of Science, University of Hong Kong. “ Misorganization of the initiation of replication can cause either a deficiency or an excessive duplication of the chromosomal DNA, resulting in breaks in DNA strands, total chromosomal rearrangements, and genome instability, which is a feature of nearly all human cancers.
Inhibition of replication initiation is an effective anti-cancer strategy for selective killing of cancer cells through apoptosis while normal human cells either stop in the G1 state (growth) or withdraw from the cell cycle to the G0 state (inactive).
Professor Chai noted, “Our studies lay a solid basis for identifying pairs of reactions, which are critical for the identification of origin and the loading of helicase, with the potential to be targets for screening and designing anticancer drugs.”
“This study demonstrates the power of interdisciplinary approaches to answer fundamental questions in the life sciences.” Commented Professor Danny Leung, Associate Professor in the Department of Life Sciences, HKUST, and Director of the HKUST Center for Genetics Research. Prof. Leung’s team was responsible for the epigenomic analyzes and bioinformatics for this study. The center coordinates the efforts of the Epigenetics Project in Hong Kong and facilitates regional researchers in conducting epigenomics studies.
“It started with the atomic model of the ORC yeast attached to the original DNA and the discovery that a single element in one of the subunits is responsible for the base-specific recognition of the DNA by the ORC. We performed genome-level assays and biochemical experiments to determine the binding properties, which led to the model that removing This model is the basis of human ORC behavior, culminating in an insight into the evolution of ORC as eukaryotes embrace more complex genomes and epigenomic structures.This insight also holds important information about disease mutation that often correlates with the flexibility of DNA replication.
The study has been published in the journal Nature Communications On January 4, 2021.
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