DNA DAMAGE TOLERANCE IN EUKARYOTES

Cancer is one of the major causes of death in the present world. The seemingly unrelated nature of its different types has made it very difficult to find a common cause that can trigger it. However, a growing body of evidence supports the idea that the roots of cancers lie in mutations in DNA, the genetic material of cells. DNA damages caused by extrinsic or intrinsic agents are usually removed from DNA and repaired by one of the several DNA repair systems of the cell preserving the genetic information. However, high exposure to DNA damaging agents can lead to the accumulation of unrepaired DNA damages that can block the replication machinery leading to cell death. To ensure survival, cells have evolved mechanisms that can sustain DNA replication on damaged DNA. These so-called damage tolerance or DNA damage bypass processes allow replication to continue on damaged DNA without removing the damaged bases. In humans, increased error-prone bypass of DNA lesions causes increased mutagenesis and a rise in the incidence of cancers, whereas error-free replication of damaged DNA contributes to genetic stability.

In yeasts, the Rad6-Rad18 ubiquitin-conjugating complex governs three alternative pathways of replication of UV-damaged DNA: the Rad5-dependent error-free, the DNA polymerase η dependent error-free, and the DNA polymerase ζ and Rev1 dependent error-prone damage bypass.

The Rad5-dependent pathway operates through a template switching mechanism, but specialized, so-called translesion synthesis DNA polymerases work in the Polη and also in the Polζ and Rev1 dependent pathways.

To activate any of the above three sub-pathways, first PCNA, the processivity factor of the replicative polymerase has to be ubiquitinated by Rad6-Rad18. Monoubiquitination of PCNA activates the Polη and the Polζ pathways, dependent on translesion synthesis polymerases. Polyubiquitination of that same residue of PCNA activates the Rad5 pathway.

The most important questions about the Rad6-Rad18 dependent DNA lesion bypass concern its regulation: How can ubiquitination of PCNA govern all three pathways? What are the downstream steps?

Our main goal is to investigate the regulatory mechanisms of DNA lesion bypass by Rad6-Rad18.

Our hypothesis: To maintain the accurate duplication of the genetic material, the access of low fidelity translesion synthesis DNA polymerases, such as Polη and Rev1, to the replication fork must be restricted to DNA lesion sites. Also, a mechanism should exist which provides preference to error-free lesion bypass pathways over the error-prone lesion bypass pathway. The regulation most probably involves specific protein interactions and posttranslational modifications, as strongly suggested by genetic data from yeast. Proteins that interact with translesion synthesis polymerases are most probably involved in the regulation of DNA damage bypass reactions.

We will identify proteins that mediate the Rev1 dependent lesion bypass pathway and characterize these proteins by genetic and biochemical means. We will investigate whether the replicative polymerase Polδ is subject to posttranslational modification upon encountering a DNA lesion. Also, we will examine the role of PCNA in mediating protein interactions necessary for activating the switch between the three subpathways. Our results will provide insight into the mechanism of the regulation of lesion bypass pathways and the polymerase exchange reactions between the replicative polymerase and translesion synthesis polymerases occurring at DNA lesion sites.