Every human being starts out as just one fertilized egg. In adulthood, that single cell has turned into about 37 trillion cells, many of which continue to divide to create the same amount of fresh human cells every few months.
But those cells face a formidable challenge. The average division cell should copy – perfectly – 3.2 billion base pairs of DNA, about once every 24 hours. The cell’s replication machinery does this wonderfully, copying genetic material at a hurried rate of about 50 base pairs per second.
Still, that’s far too slow to duplicate the entire human genome. If the cell’s copying machines started at the end of each of its 46 chromosomes at the same time, it would produce the longest chromosome — No. 1, at 249 million base pairs — in about two months.
“The way cells naturally get around this is they start replication in multiple places,” said James Berger, a structural biologist at Johns Hopkins University School of Medicine in Baltimore, who co-authored a paper on DNA replication in eukaryotes. in the 2021 Biochemistry Annual Review. Yeast cells have hundreds of possible sources of replication, as they are called, and animals such as mice and humans have tens of thousands of them, scattered throughout their genomes.
“But that poses its own challenge,” says Berger, “which is, how do you know where to start and how do you time everything?” Without precision control, some DNA can be copied twice, causing cellular pandemonium.
It’s especially important to keep the DNA replication kickoff tight to avoid that pandemonium. Today, researchers are moving toward a full understanding of the molecular checks and balances that have evolved to ensure that each origin initiates DNA copying once, to produce exactly one completely new genome.
Do it right, do it fast
Bad things can happen if replication doesn’t start correctly. To copy DNA, the DNA double helix must open, and the resulting single strands — each of which serves as a template for building a new, second strand — are vulnerable to breakage. Or the process hangs. “You really want to solve replication quickly,” said John Diffley, a biochemist at the Francis Crick Institute in London. Problems during DNA replication can cause the genome to become disorganized, which is often a major step on the road to cancer.
Some genetic diseases also result from problems with DNA replication. For example, Meier-Gorlin syndrome, which involves short stature, small ears, and small or no kneecaps, is caused by mutations in several genes that help trigger the DNA replication process.
It takes a tightly coordinated dance involving dozens of proteins before the DNA copying machine starts replicating at the right point in the cell’s life cycle. Researchers have a pretty good idea of which proteins do what because they’ve managed to get DNA replication to take place in cell-free biological mixtures in the lab. They recreated the first critical steps in initiating replication using proteins from yeast – the same kind used to make bread and beer – and they also recreated much of the entire replication process using human versions of replication proteins .
The cell controls the start of DNA replication in a two-step process. The whole purpose of the process is to control the actions of a crucial enzyme – called a helicase – that unwinds the DNA double helix in preparation for copying it. In the first step, inactive helicases are loaded onto the DNA at the origin, where replication begins. During the second step, the helicases are activated to unwind the DNA.
Done (load the helicase)…
The process begins with a cluster of six proteins at the origin. This cluster, called ORC, is shaped like a bilayered ring with a handy notch that allows it to slide onto DNA strands, Berger’s team has found.
In baker’s yeast, a favorite for scientists studying DNA replication, these starting sites are easy to recognize: they have a specific DNA core sequence of 11 to 17 letters, rich in chemical bases of adenine and thymine. Scientists have watched ORC grab hold of the DNA and then slide along, searching for the sequence of origin until it finds the right spot.
But in humans and other complex life forms, the starting sites aren’t so clearly delineated, and it’s not entirely clear what causes the ORC to settle and take hold, says Alessandro Costa, a structural biologist at the Crick Institute who, with Diffley, wrote about the initiation of DNA replication in the 2022 Annual Review of Biochemistry. Replication seems more likely to start where the genome—normally tightly coiled around proteins called histones—has come loose.
The initiation of DNA replication begins at the end of the previous cell division and continues through the cell cycle phase known as G1. DNA synthesis takes place during S phase. Levels of a protein called CDK are critical to ensuring that DNA is replicated once and only once. When CDK levels are low, helicases can jump onto the DNA and begin unwinding it. But repeated binding does not happen because CDK levels rise, and this blocks the helicase from binding again.
Once ORC has settled onto the DNA, it attracts a second protein complex: one that includes the helicase that will eventually unwind the DNA. Costa and colleagues used electron microscopy to find out how ORC lures in first one helicase and then another. The helicases are also ring-shaped and each opens to wrap around the double-stranded DNA. Then the two helicases close again, facing each other on the DNA strands, like two beads on a string.
At first they just sit there, like cars with no gas in the tank. They have not yet been activated and for the time being the cell will continue as normal.
