Almighty Power

 

The world changed in 1953 when Watson and Crick and their chums revealed the shape of the stuff of life. Our genetic material, DNA, was made up of pairs of long, intertwined chains of nucleic acids. Whether at the time we realised it, the publication of their little paper in Nature launched the age of molecular biology.

Sketch of DNA made by Odile Crick, Francis Crick’s wife, for Watson and Crick’s 1953 paper. The two ribbons represent the phosphate sugar backbones and the horizontal rods the pairs of bases holding the chains together. The arrows show that the two chains run in opposite directions. From Watson and Crick, 1953.

A couple of years later Francis Crick gave a remarkable talk to the RNA Tie Club in Cambridge in which he outlined how the structure of DNA offered mechanisms by which essentially all the fundamental processes of life might work. If the double helix was ‘unzipped’ each strand could provide a template for a new strand to be made with a complementary sequence of base units. Thus when cells divided two identical sets of double-stranded DNA were available, one for each new daughter cell.

Deciphering the code

Pulling the two strands apart would also make them available to be copied into an intermediate (RNA) that could carry genetic information to the cellular machine that makes proteins (that we now know as a ribosome). Crick had the idea that a set of adapter molecules must exist that could pick up amino acids (the building blocks of proteins) and present them to this machine in a way that enabled the correct one out of the 20 or so in the cell to be joined to the growing protein, as specified by the DNA/RNA sequence of a gene.

All this duly came to pass in a spectacular series of discoveries.

The first, in 1958, was the experiment in which Matthew Meselson and Franklin Stahl showed that each of the two double-stranded DNA helices arising from DNA replication comprised one strand from the original helix and one newly synthesized — ‘semiconservative replication’.  John Cairns, the British physician and molecular biologist, described this as “the most beautiful experiment in biology”, as vividly recorded by Horace Judson in his wonderful account of those days, The Eighth Day of Creation.

In 1961 Sydney Brenner, François Jacob and Matthew Meselson discovered messenger RNA, the intermediate nucleic acid that carries the code of genes to the ribosome. In the same year Crick, Brenner, Leslie Barnett and R.J.Watts-Tobin showed that the genetic code uses a ‘codon’ of three nucleotide bases that corresponds to an amino acid. Marshall Nirenberg and Heinrich Matthaei deciphered the first of the 64 triplet codons and Har Gobind KhoranaRobert Holley and Marshall Nirenberg shared the 1968 Nobel Prize in Physiology or Medicine for determining the complete genetic code by which every amino acid is encoded by three base sequences of RNA.

Protein synthesis by ribosomes. Ribosomes are made up of two complexes (a large and a small subunit) of over 50 proteins and RNAs. They are present in all living cells. In the process of protein synthesis (translation) ribosomes link amino acids together in the order specified by messenger RNA (mRNA). The link is a peptide bond, hence the process is called peptide synthesis.

Ribosomes move along mRNA molecules sequentially, capturing transfer RNAs carrying the appropriate amino acids to be matched by base-pairing through the anti-codons of the tRNA with successive triplet codons in mRNA. As amino acids are linked together the protein chain grows and the tRNAs, no longer carrying amino acids, are released.

The largest protein (titin — it folds up like a molecular spring to give elasticity to muscle) has over 30,000 amino acids. Very short proteins are usually called peptides (e.g., insulin with 51 amino acids, oxytocin with 9 amino acids).

Thus were revealed the pieces of molecular lego upon which all life depends. It had all occurred in an extraordinarily short space of time after the resolution of the structure of DNA. The story goes that when Watson and Crick finally convinced themselves that they had worked out DNA they retired to The Eagle pub for lunch where Crick announced to the startled clientele that they had just discovered the Secret of Life! Francis Crick, notoriously, is not remembered for either his reticence or modesty and his outburst must have been met with shaking heads and shrugged shoulders from the assembled lunchers. However, with the benefit of hindsight, it’s hard to argue with his assessment.

Fast forward to the twenty-first century

The discoveries of the 1950s and 1960s, although revealing the heart of life, were only the beginning and the ensuing years have witnessed truly breath-taking advances in our knowledge of the living world. One can’t help wondering whether, back in those days, Crick or Brenner or some other visionary might have peered into their morning coffee and muttered “Now we know how it works, wouldn’t it be fun to be able to tinker with the genetic code — to be able to make proteins to order, so to speak.”

It’s taken a while but in the 1970s genetic engineering came into existence as a way of changing the genetic make-up of cells — knocking out genes or introducing new genes. Over the last ten years or so this has progressed to using modified viruses to boost the immune response in patients with blood cancers and to treat other diseases.

This period has also seen the emergence of genome (or gene) editing that, in contrast to genetic engineering, makes specific changes to the DNA of a cell. This uses ‘molecular scissors’ to cut DNA at a known sequence. When the cut is repaired by the cell a change or ‘edit’ is made to the sequence. The best-known method is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or CRISPR/Cas9, Cas9 referring to the enzymes that do the cutting. We summarized how CRISPR works in Re-writing the Manual of Life but it’s worth reminding ourselves that this method is yet another example of using what nature provides. It’s based on the way bacteria build their antiviral defense system by incorporating sequences of DNA fragments from previous viral infections that are used to detect and destroy DNA from similar viruses during subsequent infections.

Upping the ante

Until now gene editing has mainly been used to make single changes at one or a few points in genomic DNA. Gregory Findlay, Lea Starita, Jay Shendure and colleagues at the University of Washington in Seattle have just changed all that by, in effect, making all possible variants in the BRCA1 gene and seeing what each did to the activity of the BRCA1 protein.

The object of their attentions is a very big gene that encodes a very big protein: it has 1884 amino acids (so that’s 3 x 1884 = 5652 coding bases). Why did they pick such a tough nut for their tour de force?

The BRCA1 (breast cancer 1) protein is essential for one of the processes by which cells can repair damaged DNA. If this protein doesn’t work properly mutations may accumulate and lead to cancer, making BRCA1 a ‘tumour suppressor’. As we explained in A Taxing Inheritance, women with a faulty BRCA1 gene have a 60 – 90% lifetime risk of breast cancer and a 40 – 60% risk of ovarian cancer (compared to 12% and 1.5% in the general population, respectively), facts that have been publicized by the actress Angelina Jolie.

BRCA1 has been sequenced in millions of women and a huge number of variants in the DNA sequence have been found. As more is learned about BRCA1, ways of targeting cells with mutant forms are being developed (e.g. by an effect called synthetic lethality). But these advances highlight the problem that we simply don’t know what, if anything, most of the sequence variants do. They’re delicately called ‘variants of uncertain significance’ — science-speak for ‘we haven’t a clue.’ And until we do have a clue there are no grounds on which to decide whether a particular mutant might turn out to be harmful.

Covering all bases

To be accurate Findlay et al. didn’t quite ‘do’ all the possible variants — they just covered the regions known to be important in the actions of the protein. Even so, in applying ‘saturation genome editing’ to these key regions of BRCA1, they were able to test the effects on BRCA1 activity of 3,893 single nucleotide variants (SNVs).

 

Mind blowing. Don’t try to read the detail but this extraordinary picture represents the critical regions of BRCA1. The letters are the sequence of bases (A, C, G & T) of the normal BRCA1 gene, the vertical columns of four boxes representing the possible variants at each position. In the work by Findlay et al. every possible variant was made — almost 4,000 — and tested for activity. From Findlay et al. 2018.

 The test vehicle for this ‘shot-gun’ approach was a cell line grown in culture that can only survive if BRCA1 is working normally. The cells were infected with small circular DNA carrying CRISPR and Cas9 sequences together with a library of edited BRCA1 exon sequences (exons are the bits of gene sequence that appear in mature RNA after non-coding bits, introns, have been removed). The result was that mutant exons were inserted in many of the cells. The cells were grown for 11 days during which time some of the cells died and some survived. Deep sequencing of DNA extracted from the cells confirmed which BRCA1 mutations caused cell death.

The idea behind ‘deep sequencing.’ The term simply means sequencing the same region many times so that DNA present in small amounts (e.g., in just a few cells in the sample) is detected. 

The bottom line

From the nearly 4,000 SNVs tested by Findlay et al. just over 400 were missense mutations i.e. the change of a single base (nucleotide) changed an amino acid in the protein so that BRCA1 was disabled. A further 300 SNVs reduced protein expression. Critically, Findlay et al. identified variants already known to block BRCA1 activity, thereby confirming the accuracy of their method.

It is probable that more sophisticated models will be used in the future but it seems that these results will be immediately helpful in screening BRCA1 variants detected in breast tissue so that a rational decision can be made as to whether a given SNV represents a cancer risk or whether it is harmless.

Deluded?

One might wonder whether, now that molecular biology has reached a stage where artificial proteins can be made in the lab, its practitioners may feel they have acquired almighty power — become human manifestations of a supernatural creator of the kind Richard Dawkins demolished in the The God Delusion. “No chance” I would say, in my cheerfully self-appointed role as spokesman for the community, happily aligning myself with Dawkins’ view that belief in a personal god is merely self-delusion.

The experiments tinkering with BRCA1 are quite astonishing but, I’m sure Gregory Findlay and his friends would readily admit, they’re merely using the methods now available to ask important questions. In thinking about how natural selection works Dawkins came up with the analogy of a blind watchmaker. You might argue that twenty-first century biologists are molecular watchmakers with 20-20 vision, able to produce bespoke proteins. No supernatural forces are needed, merely the knowledge and skill to be able to tweak nature’s own machinery.

References

Watson, J.D. and Crick, F.H.C. (1953). Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid. Nature 171, 737-738.

Findlay, G.M. et al. (2018). Accurate classification of BRCA1 variants with saturation genome editing. Nature 562, 217–222.

H.F. Judson (1996). The Eighth Day of Creation: Makers of the Revolution in Biology. Cold Spring Harbor Laboratory Press, Plainview.

Crick, F. (1955). A Note for the RNA Tie Club (Speech). Cambridge, England. Archived from the original (PDF) on 1 October 2008.

Meselson, M and Stahl, F.W. (1958). The replication of DNA in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. 44, 671–82.

Brenner, S., Jacob, F. and Meselson, M. (1961). An unstable intermediate carrying information from genes to ribosomes for protein synthesis. Nature 190, 576-581.

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