DNA replication and repair in vertebrate cells

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Published: 23 Jul 2015
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Dr Vincenzo Costanzo - DNA Metabolism research program, IFOM, Milan, Italy

Dr Costanzo talks to ecancertv at IFOM EMBL about vertebrate genome stability, focusing on the DNA replication and repair in vertebrate cells.

Additionally he discusses his research using extracts derived from Xenopus laevis eggs, which allow extensive biochemical analysis which can be reproduced in vitro complex cell cycle events such as chromatin formation, nuclear assembly, semi-conservative DNA replication, chromosome assembly and mitotic spindle formation.

We basically work on aspects related to DNA repair and DNA replication in vertebrate cells.

This is highly significant for understanding how cancer develops and how cancer basically arises in humans.

Basically what happens is that in cancer cells it has been found many years ago that DNA is not stable.

This means that the replication and the repair of the DNA is somehow different from normal cells.

Indeed, if you make a picture of the DNA and, in this case, if you make a picture of the chromosomes contained in a cancer cell they look really different from human normal cells because what happens is that many of these chromosomes look abnormal – there are increased numbers or there is a structural abnormality.

This underlines a very important concept that not only genes are mutated in cancer and altered but also their position on the chromosomes is not like the position that you would find in normal cells.

This is important because if a particular gene loses a particular position on chromosomes it loses the way it is normally regulated.

In cancer cells what happens is that sometimes chromosomes fuse together or break and are repaired with pieces that do not belong to them.

So we are trying to understand what happens and what are the problems that lead to this kind of DNA instability in cancer cells.

In particular we are focussing on genes that are commonly mutated in cancer cells like, for example, the BRCA2 or RAD51 gene family.

In BRCA2 mutations that lead to partial loss of these genes predispose to breast cancer and this is highly frequent because 5% of women affected by breast cancer have a familial type of breast cancer and in this familial type of breast cancer BRCA2 is highly mutated.

So how do we study BRCA2?

BRCA2 is a very large gene and consequently gives rise to a very large protein.

This gene is essential, meaning that if you completely knock it out, like geneticists will do in order to understand the function, you lose the survival of the cell.

This means that cells are very sensitive to complete loss of BRCA2.

But this is a problem to study BRCA2 because if we don’t have a cellular model then we cannot study, we cannot delete BRCA2 otherwise the cell will die.

How do we overcome this problem?

So we decided to use another system in which BRCA2 is highly analogous to the human BRCA2 gene.

In this case this system relies on Xenopus laevis eggs.

So the Xenopus laevis is a vertebrate, is a frog, that produces eggs that are a very large unicellular, basically, system where you can easily deplete using an antibody the protein of interest.

In what we do in the lab, we make an extract out of these eggs and we deplete BRCA2 out of the extract and we study how DNA is replicated and repaired when this protein is not around.

We recently found that when BRCA2 is not around the DNA is full of gaps – what does it mean?

It means that instead of having a normal helical double strand structure it has pieces of region where the DNA is single stranded.

This means that the DNA has not been perfectly duplicated and this is the first time that this has been shown.

In particular this is very important because a recent drug, namely the PARP inhibitor, which blocks the action of PARP1 which is an enzyme deputised to the repair of single strand gaps, has been shown to be very effective in breast cancer.

So what we think is happening is that BRCA2 is important to repair single strand DNA gaps and when BRCA2 is not around these gaps accumulate.

Normally cells are able to cope with this and to repair these gaps by an alternative pathway and this alternative pathway relies on PARP1.

But when you block PARP1 this alternative pathway does not work, therefore there is a synergistic effect that kills specifically cells in which BRCA2 doesn’t work.

So this is the first example whereby knowledge on the DNA repair function has been linked somehow to a drug that inhibits a repair function that can specifically target cancer cells and not normal cells.

This is very different from chemotherapy that basically does not discriminate between normal cells and cancer cells.

Therefore, the idea for the future is to understand even better how the fine mechanism of DNA repair works in normal cells and in cancer cells using a model system like Xenopus laevis and other systems like cells derived from cancer cell lines.

In this way we could understand if other factors contribute to the repair in these specific cancers and to see if this factor can be selectively targeted in order to build knowledge and to propose strategies to create new drugs that then can be used in the future to selectively kill cancer and non-normal cells.

So the idea is to evolve towards a selective treatment of cancer cells.

We know that this is in line with what is called precision medicine which is basically how the cancer is going to be treated in the future.

Because now that we can very rapidly have information about a number of genes mutated in a single patient we can rapidly know what is the genomic and the genetic background in these patients so that we can administer selective types of therapies in order to treat this patient with high specificity and less toxicity.

Of course, we will need to know which genes need to be targeted and we will need drugs to specifically target this.

This will be a long-term goal but we have now all the knowledge and all the tools set in order to reach this kind of knowledge. So now we think we understand what cancer is.

The problem is now how to treat it and that’s what we are trying to do.