Understanding the process of DNA replication

Bruce Stillman, Oliver R. Grace Professor and CEO of Cold Spring Harbor Laboratory, traces the history of research on genetic inheritance.

Bruce Stillman

Biologist and biochemist

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Bruce Stillman

Biologist and biochemist

02 Jul 2021

Key Points

The research of 19th century pioneers like Charles Darwin and Gregor Mendel laid the groundwork for modern discoveries, such as the structure of DNA.
Watson and Crick’s double helix didn’t tell us how the DNA is copied, which is what biologists like Bruce Stillman are working on now.
Scientists have unlocked the process to such an extent that they can now reconstitute chromosome replication in a test tube.

The giants of 19th century biology

The history of inheritance traces back to Charles Darwin, who proposed the theory of natural selection as the way in which species evolve from other species. This sets the stage for understanding all of biology.

In 1859, Darwin published his famous book, On the Origin of Species. In 1865, Gregor Mendel, who didn’t know Darwin, published his papers on the genetics of peas. Mendel showed that there are quantitative traits that are inherited from one generation to the next and discovered recessive and dominant genes. This led to the concept of an inherited trait, or what we now call a gene.

Later, and again, completely independently, the Swiss physician Friedrich Miescher isolated from blood what he called nuclein, which we now call DNA. So, we can see that the second half of the 19th century set the stage for what became the understanding of inheritance.

A brief history of DNA

Photo by cooperr

In the early part of the 20th century, Thomas Hunt Morgan and his colleagues demonstrated that mutations – the type of traits that Mendel saw – also were localised on chromosomes.

The really big breakthrough was in 1953, the year of my birth, when Watson and Crick, and Franklin and Wilkins, discovered the structure of the double helix. That led to several fundamental new understandings, such as the self-templating, or copying, of the strands of DNA so that there could be a mechanism for inheritance. The line of base pairs along the DNA could alter to generate mutations, and the sequence of those base pairs created a genetic code for proteins, as well as for other molecules and cells.

That’s a very brief history of DNA – except that Watson and Crick’s double helix didn’t tell us how the DNA is actually copied, which is what I’ve been working on.

A million kilometres of DNA per minute

We have two metres of DNA inside each of our cells. If you stretch out the DNA from a single cell, it’ll stretch for two metres. One metre comes from mum, one from dad.

In human cells, this DNA is distributed between 46 chromosomes, which have to be accurately duplicated before cells divide. Once a fertilised egg, which is how we start life, is produced, that creates a cell division: one cell becomes two, two become four, four become eight and so on, to produce an organism of (in our case) many hundreds of trillions of cells. Just in our bone marrow alone, we have five hundred million cells born every single minute of our life. Five hundred million!

If you take the DNA for each cell, that’s a million kilometres of DNA being made every minute in the bone marrow alone – and a million kilometres is a long, long way. I’ve asked a lot of people how far they think it is. To give you a sense, it’s 25 times around the equator of the Earth. So just in our cells, in the bone marrow, we’re making this much DNA all the time throughout our lives – and that’s only one proliferative tissue; we have many others. The lining of our gut, the hair follicles, the skin and all the other proliferating tissues of our body are producing massive amounts of DNA.

This process occurs in a very regulated way. Cells are controlled in their decision whether to proliferate or not, whether to divide or not. This is a normal control process that takes into account all of the environmental effects.

Learning from yeast

Photo by Rattiya Thongdumhyu

I’ve been studying the process of DNA replication for my entire career and have been using different organisms that provide different advantages for studying this process. One of them, of course, has been human cells, because we’re fundamentally interested in how human chromosomes are duplicated. However, the real workhorse has been budding yeast – the same type of yeast that is used to make beer, bread and wine.

Yeast has been a phenomenally useful tool for understanding cell biology. What’s interesting about this is that humans and budding yeast evolved, or separated from each other from a common ancestor in evolution, about a billion years ago. That’s a long time. Yet, about 23% of the genes in yeast are very similar to the genes in human cells. In fact, it is possible to take human genes, put them into yeast and have them work and keep the yeast alive. Over a billion years of evolution, the process of DNA has accurately copied this template so that a yeast gene now or a human gene now can be put into yeast. Of course, alongside that is the strong selection during evolution for the function of these genes, but unless you have an accurate copying machine, that selection is not going to work very well.

This really sets the stage for understanding the process of DNA replication. What happens during that process is that mutations occur. These mutations, including during the production of sperm, can lead to diseases such as cancer and autism.

All cancer cells are different

During the process of copying DNA and the 46 chromosomes in our cells, and prior to the separation of those chromosomes, errors can occur. These are pre-existing mutations, and they cause what we now think of as Darwinian selection.

Some of those mutations can lead to disease. Particularly in the case of cancer, these mutations can occur in proteins that are responsible for copying the DNA itself, or repairing the DNA if mistakes are made during the copying process. If the genes that are affected by mutation are those used in the copying or repair processes themselves, that can lead to more mutations inside proliferating cells like cancer cells.

As a consequence, all cancer cells are different. Of course, breast cancer is different from prostate cancer, which is different from lung cancer, because it arises in a different tissue. But within those individual tumour types, there are many subtypes of cancers that are caused by the different types of mutations in genes. Even within a single clone of a cancer, individual cells can have different genetic mutations because of the continuing production of mutations during their proliferation. This is one of the reasons why cancer is so difficult to treat. You’re constantly spinning off resistance to the therapies that we can devise to fight cancer.

An exquisitely accurate copying machine

Photo by Desing_Cells

The heterogeneity in cancer is extraordinary, but what’s more extraordinary is that we all don’t get massive amounts of cancer in our body.

That’s because the copy machine and the associated repair of the mistakes that are made during the copying process are exquisitely accurate. There are only a few mutations per billion base pairs of DNA duplicated inside a cell. And since only a small percentage of our genome is copied into protein, for the most part, these random mistakes tend not to hit the function of proteins.

Also, we have an immune system that surveils the proliferating cells and can get rid of cells that produce new proteins in our cells because of mutations. This is one of the reasons why we don’t get more cancer than we do.

From theory to proof

Each of our chromosomes contains one large double helix. Watson and Crick realised the implications of how this templating mechanism worked, but it was a theoretical model. It was only later, in 1958, that it was proven to exist by Meselson and Stahl in a famous experiment. They demonstrated that the double helix does separate and is self-templated to produce two new strands. Each new double helix has one old strand and one new strand.

However, the enzymatic machinery, or how this entire process occurs, was not known at that time. In my career, I’ve focused on how the more complicated genomes of what we call eukaryotes – cells such as human cells that have a nucleus with many chromosomes inside them – duplicate and how they’re copied. That process is fundamental to understanding the control of the cell division cycle: how cells divide, how cells commit to cell division and how this process produces accurate, duplicated chromosomes.

We’ve discovered an entire machinery inside the cells that recognises how the DNA is going to start to be copied, how it is committed to be duplicated throughout the cell division cycle and how the enzymatic machinery separates and copies the double helix. We’ve discovered all of the enzymes, so now we can reconstitute this entire process of chromosome replication in a test tube, using entirely purified proteins whose workings we understand.