Bypass of DNA damage
We are interested in how proliferating higher eukaryotic cells cope with DNA damage. We investigate the cellular mechanisms that make it possible to replicate damaged DNA, in order to understand how defects in these damage bypass mechanisms affect mutagenesis and cell survival.

A need for DNA damage bypass

DNA lesions arise continuously due to endogenous or environmental agents, and may also be elicited by chemotherapeutic treatment. In proliferating cells some of these lesions are inevitably encountered during DNA replication. Stalled replication can lead to incompletely duplicated chromosomes, cell cycle arrest and cell death. Therefore several mechanisms exist that enable the replication machinery to bypass sites of damaged DNA. Successful bypass of lesions allows the cell to survive, but it is the main generator of mutations.

Damage bypass mechanisms

Bypass mechanisms
Figure 1
 The conceptually simplest procedure of bypassing lesions encountered during DNA replication is translesion synthesis, whereby the accurate and damage-intolerant replicative DNA polymerase is temporarily replaced by a specialist polymerase that can synthesise the new DNA strand across the site of damage. This process, while not always mutagenic, is inherently error-prone. Entirely error-free bypass of DNA lesions is only possible by replicating the damaged section using an alternative, undamaged template. One known mechanism capable of achieving this is homologous recombination, which involves breaking the damaged DNA strand and extending the broken ends along strands of the replicated sister chromatid. In addition, the existence of a genetically distinct error-free bypass pathway has been demonstrated in bacteria and yeast. This pathway is thought to operate by switching template to the homologous, newly synthesised strand of the sister chromatid.

Experimental approaches

We are investigating the full range of higher eukaryotic DNA damage bypass processes and their interactions. For the time being, the workhorse of our studies is the DT40 chicken cell line, which is well established as the genetically most amenable vertebrate system. Using a variety of DNA damaging agents as well as replicating shuttle plasmids containing synthetic DNA lesions, we are dissecting the contribution of individual genes to different aspects of DNA damage bypass and mutagenesis.