DNA replication and human diseases
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We are pursuing research into DNA replication in eukaryotes, as well as its potential uses in the treatment of replication-related human diseases.
DNA replication is one of the most fundamental cellular processes that govern the billions of cell divisions from a single fertilized egg to develop into a full-grown human. Every single step of the DNA replication process is strictly controlled, ensuring the genome is faithfully duplicated per cell division. When errors occur, catastrophic consequences are expected because the mistakes would be amplified and accumulated through DNA replication during the development process. It is not surprising that DNA replication intimately correlates with human diseases, such as such as cancer, aging, and genetic disorders.
How is chromosomal DNA replicated in eukaryotes?
In eukaryotic cells, DNA replication is initiated from multiple origins along each chromosome . The activities of each origin are tightly regulated to maintain genome integrity. Mis-regulation of replication initiation can cause either under- or over-replication of chromosomal DNA, which will induce DNA strand breaks, gross chromosomal rearrangements, and genome instability, a characteristic of almost all human cancers. Inhibition of replication initiation is considered as an effective anti-cancer strategy for the selective killing of cancer cells through apoptosis while normal human cells arrest in G1 or withdraw from the cell cycle into G0 state .
DNA replication is initiated from each replication origin no more than once per cell cycle. To achieve this regulation, replication licensing is separated temporally from origin firing through oscillating kinase activities during the cell division cycle.
At early G1 phase, when kinase activities are reduced to a low level, replication origins are reset to a state competent for replication licensing. Subsequently, origin recognition complex (ORC) is able to work with Cdc6 and Cdt1 to assemble Mcm2-7 complexes, the catalytic core of the replicative helicase, onto origin DNA as a head-to-head double hexamer (DH) encircling dsDNA. Throughout the remaining G1 phase, the Mcm2-7 DH remains inactive in its helicase activity but it is poised to be activated.
Molecular Biology of the Cell, 5th ed.
Upon entering S phase, the rise of kinase activities drives origin firing to initiate DNA replication. Two kinases, DDK (Dbf4 dependent kinase Cdc7) and S-CDKs (S-cyclin dependent kinases), act in concert with various origin-firing factors to convert the Mcm2-7 DH into two active Cdc45-Mcm2-7-GINS (CMG) helicases. During this process, the Mcm2-7 DH encircling the duplex DNA must reconfigure in multiple steps in order to trigger initial DNA melting for dsDNA separation, to uncouple the DH into single rings, to exclude the lagging strand DNA from each MCM ring, and to capture and encircle opposite strands of the duplex DNA for bidirectional replication. Currently, the detailed mechanisms underlying the events leading to replication initiation are not well understood at a molecular level.
A large number of accessory factors are also required to facilitate fork progression along chromatin DNA and maintain genome stability. Under normal conditions, the replisome can travel hundreds of kilobases without falling off while ploughing through DNA wrapped in histone octamers, known as nucleosomes. It has been recently shown that the replisome core components and some accessory factors work together to recycle parental histones and restore epigenetic marks as DNA is being replicated. During fork progression, the replisome also encounters transcription complexes and other types of tight protein-DNA complexes. In certain situations, these roadblocks, if not properly removed, may become an impediment to replication resulting in fork stalling and even chromosome instability. Under stress conditions, stalling forks send out stress signals to remodel replisomes, among other targets, to temporarily arrest DNA replication until DNA damage is repaired and conditions return to normal. Obviously, the regulation of the eukaryotic replisomes in vivo is far more complicated than its counterparts in prokaryotes.
In eukaryotes, chromosomal DNA is organized into chromatin with nucleosome as the basic repeating unit comprising ~147 bp of duplex DNA, wrapped around two H2A-H2B dimers and one H3-H4 tetramer2. The epigenetic information in the form of post-translational modifications of histones further define diverse functional states of chromatin to impose precise control upon transcriptional programs, which are closely linked to cell differentiation and cell reprogramming. The relevant epigenetic marks are transmittable to progenies through cell division cycle. The relevant epigenetic marks are transmittable to progenies through cell division cycle. During DNA replication, parental nucleosomes bearing epigenetic marks are disrupted ahead of each replication fork to allow duplex DNA unwinding and subsequent DNA synthesis. To maintain the fidelity of epigenetic landscape, the evicted parental histones have to be recycled and deposited at almost the same position on the newly synthesized strands. Although DNA replication has been extensively investigated, how epigenetic information encoded by histone modifications is inherited through chromatin replication remains enigmatic.
Most of our knowledge about the regulation of DNA replication in eukaryotes comes primarily from yeast studies. Although general mechanisms obtained from yeast could be applied to human cells, the detailed regulation of this process could be quite different between yeast and human and may be relevant only to their respective species. We currently exploit biochemistry, structural biology, genomics, and chemical biology approaches to investigate the structure and function of the protein complexes involved in DNA replication from both yeast (Nature 2015; NSMB 2017; Mol Cell 2017; Nature 2018; Nat Commun 2021, 2022) and human (Cell Discov 2021; Cell 2023), epigenetic control (Science 2023), and disease related organisms. We aim to elucidate the detailed mechanisms regulating DNA replication to provide a structural framework for understanding the pathogenesis of replication related diseases and identifying potential strategies for their effective therapeutics.