Repair of replication-associated double strand breaks

The accurate inheritance of the genetic material (DNA) is key to the survival of all organisms. DNA has to be copied (replicated) and then divided into two identical halves which separate at cell division into the two daughter cells, ensuring that the daughter cells inherit identical copies of the genome. We want to understand how cells repair DNA breaks that occur during replication and how repair is coordinated with replication.

DNA double-strand breaks (DSB) are dangerous forms of DNA damage. A single unrepaired DSB is can be lethal to a cell and inaccurate repair of a DSB can result in mutations or chromosome rearrangements. Cells are particularly vulnerable to DSBs that occur during replication. The break has to be repaired and replication restarted as it is vital that all of the genome is copied. Restart can be at the expense of an increased error rate leading to mutations and an increase in genome rearrangements. Since such changes can lead to cancer it is vital that copying is coordinated with repair and restarts correctly.

Essentially all mechanistic models for DSB repair look at a single DSB in a linear molecule, ie it has two ends. In S phase breaks have one end. Here we have set up a system in fission yeast where we can induce a break during replication at a specific place in the genome. This enables us to study the events involved in repair and restart. We use fission yeast to study how DNA repair is coordinated to ensure copying restarts correctly because it is easy and cheap to manipulate and most importantly due to the biology of fission yeast we can induce a specific DNA break during replication. This unique system means that we can study repair and replication restart at a particular site in detail and this cannot be done easily in other systems. Since yeast cells repair and copy their DNA in a similar way to human cells we can use this system to model how human cells repair breaks associated with the copying of the DNA.

This information will be important and provides opportunities to rationally design novel cancer therapies. The aim of such therapies is to specifically target tumour cells. Since these are actively growing and often defective in DNA damage responses a knowledge of the processes involved in the repair of DNA breaks in S phase will in the long term inform the choice of therapeutic agents.

Cells are particularly vulnerable to DNA damage during S phase. In order to study how replication-associated DSBs are repaired we have set up a novel system in fission yeast to study site-specific strand-specific DSB repair in S phase. This system is unique as, unlike the widely-used HO and I-Sce1 endonuclease generated DSBs, it breaks only one sister chromatid and only once. We will use this to define the mechanism of repair of a site-specific one-ended DSB in S phase, either in the absence of an incoming fork or when a converging fork can rescue it to form a two-ended DSB.

One-ended DSBs can only be repaired by homologous recombination (HR). In mammalian cells non-homologous recombination (NHEJ) is the major pathway for repair but HR is essential for S phase progression. It is assumed that the critical lesion is the one-ended DSB but mammalian cells fire dormant origins of replication in response to problems in S phase and thus in most cases the one-ended break will be rescued by the converging fork. This analysis will test whether the converted two-ended DSB also requires HR. We will define whether a converged fork two-ended DSB can be repaired by NHEJ or whether prior processing of the one-ended DSB commits to HR. We will define the requirements for this processing and for resolution of HR and whether the mechanisms for repair in S phase are similar to those at endonuclease generated DSBs.

Overall we aim to establish how replication-associated DSBs are repaired, shedding light on the balance of break repair pathways in S-phase and their potential roles in the generation of genome disorders and cancer in humans.

This proposal seeks to understand how replication-associated DSBs are repaired. Beneficiaries will include those with interests in DNA repair and homologous recombination and particularly those interested in repair pathway choice and the integration of metabolic processes within cells. Researchers interested in mechanisms of replication and replication restart will also benefit, given the importance of ensuring the completion of replication.

This analysis will benefit academics interested in the link between genome stability and human disease. Thus this basic research will inform the wider DNA repair field and provide mechanistic insight into the development of genomic disorders and the accumulation of mutations during carcinogenesis. This will impact on scientists and clinicians with interests in these areas.

Since DNA replication and repair are both important targets for cytotoxic agents this project will contribute to rational design and development of novel anti-cancer drugs and/or combination therapies. Thus, contributing to the health and wellbeing of the nation and benefiting pharmaceutical companies involved in the drug development.

The skills learnt by the research workers during this project will contribute to their employability in both academia and industry. The UK economy will benefit from the training of a skilled workforce, enhancing international competitiveness in technologically advanced industries. This will contribute to the economic well-being of the nation. Public engagement will contribute to the public understanding of scientific and health care issues and hopefully inspire the next generation of young scientists, again contributing to the generation of a skilled workforce.