HKU biologists and their collaborators reveal how DNA unzipping machines work, providing clues for cancer treatment

Human life begins with a fertilized egg
in the mother's womb.
This egg multiplies through cell division and develops into our multicellular body
.
During each cell division, our genomic DNA, the blueprint of genetic information, is accurately replicated
.
Each cell carries about 2 meters of DNA and makes up 23 pairs of chromosomes
.
During our lifetime (about 70 years old), our bodies will synthesize DNA
more than a light-year long, about 10 to¹⁶ meters.
The replication process first involves melting the DNA double-strands and then separating them into two single-stranded templates for DNA polymerase synthesis into complement strands
.
Any improper regulation of this process can lead to dire consequences such as tumorigenesis and hereditary genetic diseases
.

"Uncovering the secrets of DNA replication is key to
understanding the mysteries of life.
Dr Yuanliang ZHAI, Assistant Professor in the School of Biological Sciences at the University of Hong Kong, said, "Figuring out the structure of replication machines is crucial
to understanding their molecular function (seeing is believing).
”

Since James Watson and Francis Crick determined the structure of DNA in 1953, how the double-stranded structure of DNA initially melted has been a perennial question
for biologists.
In eukaryotes, the enzyme responsible for untying the DNA double-strands during replication was first identified by our collaborator, Professor Bik-Kwoon TYE of Cornell University, in 1983 from the microchromosome maintenance protein complex (MCM) gene
in winemaking yeast.
The products of six MCM genes, MCM2 to MCM7 (MCM2-7), form a hexasubunit ring complex that acts as the catalytic core of the hydrolysis compressor, DNA replication helicase
.
In cells, in order to initiate DNA replication, the MCM2-7 complex must first assemble into a head-to-head bihexamer (DH), a double-stranded DNA
that surrounds thousands of locations on each chromosome.
Of the massively assembled MCM2-7 DHs library, only a subset of them will eventually be selected and converted into a powerful replication helicase
for DNA unwind.
It is thought that MCM2-7 DH can directly destabilize DNA, triggering the initial opening
of double-stranded DNA.
However, the underlying mechanisms remain largely unknown
.

To solve this problem, the research team tried to use cryo-electron microscopy to visualize the atomic details of MCM2-7 DH, which are almost millions
smaller than the resolution limit of the human eye.

In 2015, the team solved the cryo-EM structure
of the first MCM2-7 DH isolated from yeast 3.
8Å.
Unfortunately, the captured DNA was unstable and failed to inform the double-stranded status of the DNA bound by MCM2-7 DH
.
Recently, researchers successfully purified MCM2-7 DH from cultured human cells and determined its structure to be 2.
59Å
.
This high-resolution structure clearly demonstrates how the MCM2-7 complex destabilizes DNA, resulting in an initial opening of the DNA double-stranded at the junction of two coupled MCM2-7 hexamers
.
The team also found that MCM2-7 DHs are loaded onto DNA at tens of thousands of sites in the human genome that are mutually exclusive
with active transcription sites.
In addition, when this initial open structure is disturbed, MCM2-7 DHs can no longer assemble onto DNA, resulting in complete inhibition of DNA replication initiation
.

Dr Shangyu DANG, Assistant Professor in the Division of Life Sciences at HKUST, said: "The atomic-resolution cryo-electron microscope structure allows us to see directly the initial DNA melt, which is critical
for our understanding of the molecular mechanisms underlying DNA replication.
This study also demonstrates the importance of
collaboration.
The efforts of research groups with complementary expertise are needed to answer fundamental biological questions
.
”

DNA replication has been targeted by several drugs to treat cancer
.
However, existing drugs indiscriminately kill all cells that are dividing, as both normal and cancer cells must copy their DNA in order to proliferate
.
Therefore, the specificity of these drugs raises serious concerns
about these anti-cancer chemochemies.
A more ideal alternative is to inhibit DNA replication initiation, allowing normal cells to be stopped in the G1 phase (first growth phase) or exit from the cell cycle to the G0 state (resting phase).

But cancer cells undergo apoptosis
.
Therefore, inhibition of replication initiation can be used as a novel and effective anti-cancer strategy with the potential to
selectively kill cancer cells.
Our findings in this study provide high-resolution structural and mechanistic information about human pre-initiation complexes that can be used to develop non-toxic anticancer drugs
targeting MCM2-7 complexes in the future.