Derailing DNA

DNA acts like a bi-directional rail track with two types of train moving along the rails – Professor Panos Soultanas tells us more

The first complete human genome sequence was published with great fanfare more than 10 years ago and the news of this “blue print of life” generated considerable excitement amongst the research community and general public alike. Whilst the enormity and importance of the breakthrough could not be in doubt, there is still work to do to layer this basic information with the subtleties and caveats of gene expression and genome replication that give us the huge variety of cells and tissues that make up a human being as well as our amazing ability to respond to environmental and nutritional stimuli and deal with stress, disease, and the challenges of development.

The environmental context of a cell is always changing and whilst many of these changes can be fairly subtle, they still require continuous interplay of proteins carrying out different functions within the genome. There are many proteins working in consort to effect exquisite control of gene expression and genome replication. They alter chromatin structure, act as transcription factors, silence regions of the genome, proof read, repair damage, separate strands of DNA, and play many other critical roles.

This very active environment can throw up conflicts that put genome stability in peril. Sometimes the working lives of proteins within the genome can be truly treacherous and not least for those that are involved in the interplay between gene expression, DNA replication and the prevention of DNA damage. At times the proteins carrying out replication may collide with those involved in transcription and we have recently made a discovery that shows that replication is even more hazardous for a cell than first thought.

DNA replication and translation both require complex groups of proteins that act as machines and move along the genome. Consider DNA as a bi-directional rail track with two types of train: a big fast one like an eight-carriage cross country train and a small slow one like a two-carriage regional train. As it travels, the big train – the DNA replisome – is responsible for copying the DNA e.g. when a cell is preparing to divide. And the small train – the RNA polymerase – makes its journey to deal with the expression of genes contained within the DNA sequence.

Because the DNA replisome and RNA polymerase can often move in opposite directions along the same DNA lattice, head-on collisions are fairly inevitable. Just like trains, head-on collisions between proteins moving along a strand of DNA can be catastrophic and this is one reason why regions of the genome that are being transcribed often are particularly prone to damage.

In bacterial genomes, these head-on collisions present such a disadvantage that through evolution, mechanisms have arisen to ensure that the replisome is most often moving in the same direction to RNA polymerases in regions of the genome that are highly expressed or near the replication origin. These co-directional proteins can still meet, particularly considering that replication is an order of magnitude faster than transcription. This leads to the replisome catching up to the slow-moving RNA polymerases and thus the potential to crash is still high.

But until now it was thought that only head-on collisions between the DNA replisome and the RNA polymerase could lead to serious DNA damage. Reason being that we thought that if the fast and slow proteins meet going in the same direction along the track then the faster DNA replisome just slows down and follows along behind the RNA polymerase until it has finished its job and moved out of the way. Our new research shows that this isn't the case at all and in fact they do collide quite often causing what, in this analogy, we could only describe as a major derailment!

This exacerbates the problem in areas of high transcription where there are many RNA polymerases working on genes that are in high use. For example, in bacteria that are in a phase of rapid growth, ribosomal RNA genes are very highly expressed in order to ensure the rate of protein synthesis keeps up with demand. There may even be multiple RNA polymerases working in tandem to generate sufficient ribosomal RNA. This increase in transcription, as well as spontaneous pausing events, can cause chaos. In particular, in this scenario the RNA polymerases are acting as if they are a lot of slow moving – or even broken down – trains running close together on the track. A fast moving DNA replisome is faced with an unimaginably huge obstacle and any failure to safely negotiate the chaos could easily result in significant copying errors.

The DNA replisome does sometimes fall off the genome but all is not lost. There are proteins called "restart replication proteins" that come in to help get it back on track. The exact method of restarting replication can sometimes depend on what has gone wrong to stop it in the first place, but this is an area of considerable uncertainty at present. There are currently two mechanisms that have been investigated in some detail in E.coli. One is mediated by a protein called PriA, which restarts replication in situations where there is a gap on just the lagging DNA strand. PriA stabilises the replication fork and prevents the part that is yet to be replicated from unwinding. PriA only works properly in the presence of another protein called RecG and mutations that prevent these two from working together can lead to PriA abortive unwinding of stalled replication forks. The other mechanism is mediated by a protein called PriC, which operates only when there is a gap on the leading DNA strand.

Although these mechanisms ensure that DNA replication can continue, they can potentially increase the risk of mistakes occurring during the copying process, particularly if such restart replication proteins are malfunctioning. In some cases these mistakes can lead to problems e.g. if the mistake causes a genetic malfunction that can lead to a cancer developing.

Another way of dealing with these conflicts between replication and transcription is to sweep the path ahead of the replication fork clear. This often involves removing a stalled transcript and restarting the RNA polymerase to send it on its way, or even sending another protein along the DNA to give the RNA polymerase a nudge. Sometimes an RNA polymerase can be stopped part way through transcription, freeing it up to continue away from the region being replicated and thus clearing the way.

So, in a developing and dividing cell, the hive of activity within the genome can raise some really significant challenges. How our molecular biology has evolved to deal with these challenges gives us an idea of the very subtle interplay between different molecular mechanisms. It is only by understanding these mechanisms that we can begin to appreciate the complexity of what it is to be a human being. The human genome sequence gives us the basic information that is required but the many layers of interpretation of that information are what gives us, and other complex organisms, our extraordinary variety and versatility.

Professor Douglas Kell, Chief Executive, BBSRC said: "This is an excellent achievement. Biological sciences as a discipline is unique because there are a collection of key ideas, tools, techniques and processes that are applied across an enormous range of topics. The interplay between gene expression, DNA replication and the prevention of DNA damage is an example of just such a tenet of biology and so this result has the potential to touch on research right across BBSRC's portfolio and beyond."

Author: Professor Panos Soultanas