Homologous Recombination at Human Centromeres: Friend or Foe?

DNA contains the full set of instructions that cells need to function correctly. In most cases, human cells carry 46 long DNA molecules, each of which is packed into a structure called a chromosome. When a cell divides into two daughter cells, the 46 chromosomes are duplicated, and it is essential that the two daughter cells receive a copy of each. If this separation process fails, daughter cells may die, malfunction, or even become harmful, leading to various human diseases, most notably cancers.

The centromere is a unique and large region found in each chromosome, known to play an essential role in ensuring that chromosomes are separated into the two daughter cells correctly. Centromeres are made of highly repetitive DNA sequences, but we still do not know why the centromere contains such repeats and why this is important in ensuring that chromosomes get separated correctly. Studying this problem will help us understand how cells ensure that this separation process is accurate, and may suggest new ways to treat cancers that are caused by centromere defects.

My research group has a long-standing interest in a process called homologous recombination (HR). HR allows cells to fix breaks in their DNA, using similar sequences as a template. Occasionally though, HR can make mistakes, resulting in additional harmful damage. We know that HR happens at centromeres, but it is unclear whether this has a helpful or a harmful effect on centromeres and the correct separation of chromosomes. Our preliminary results suggest it may be the former.

The goal of this project is to understand how HR assists centromere function, and how the proteins involved in HR prevent potentially harmful consequences. We will do this by addressing three questions:

1. How does the central mediator of HR, called RAD51, act at human centromeres? This will help us understand in detail which properties of RAD51 are critical to normal centromere function.
2. How do the modulators of RAD51 act at human centromeres? We will focus on understanding the role of two proteins called BRCA2 and PALB2, because defects in these proteins increase the risk of developing cancers. This will help us understand how BRCA2 and PALB2 prevent cancer development.
3. How are the harmful effects of HR at centromeres prevented? Understanding this mechanism will help us predict the likelihood of cancer development.

Our team has the expertise, equipment and collaborators that will allow us to answer these questions. We will be able to understand better how HR acts at centromeres and how it protects cells from the chromosome losses or gains that drive cancer development. Our research may also lead us to identify new ways to diagnose cancer and develop new therapeutics that can be used in combination with existing treatments to provide better outcomes for patients.

Homologous recombination (HR), which is catalysed by the RAD51 recombinase, plays pivotal roles in preventing human diseases associated with genome instability, including cancer, developmental disorders and premature ageing. Indeed, malfunctions of key RAD51 regulators BRCA2 and PALB2 increase the risks of these diseases. However, HR can also be an entry point for genome instability and hence must be tightly regulated. This is particularly important at highly repetitive genome regions. Among them, centromeres are especially unusual genome regions, consisting of long stretches of repeats and playing a central role in chromosome segregation. It remains enigmatic how HR acts positively or negatively at human centromeres.

Our previous work revealed that RAD51 maintains centromere integrity and assists accurate chromosome segregation in mitosis. Building on these findings, we have developed a versatile methodology to detect DNA damage at human centromeres in single cells. This technological breakthrough enabled us to demonstrate that RAD51 protects centromeres from DNA damage even in non-dividing and non-cancerous human cells. We hypothesise that RAD51 ensures the integrity of centromeres, which are susceptible to spontaneous DNA break, while also preventing harmful recombination of their repetitive sequences and, in this way, promotes genome stability.

This project will examine: i) how RAD51 protects human centromeres from DNA breakage; ii) how RAD51 is recruited to centromeres; and iii) the mechanisms that lead to the potentially harmful outcomes of HR. The use of light imaging approaches directly detecting DNA damage at centromeres, genetic tools, unbiased genome-wide sequencing technology and quantitative proteomics will be key to examine the role of HR at human centromeres, the main focus of this study. The proposed research represents an important first step towards the development of new treatments to prevent diseases associated with centromere dysfunction.