REPLICATION OF DAMAGED DNA IN YEAST AND HUMAN CELLS: IMPLICATIONS FOR MUTAGENESIS AND CARCINOGENESIS

The stalling of DNA replication machinery that occurs as a consequence of encountering DNA damage is a challenging problem for cells. To rescue the stalled replication fork, different DNA damage bypass mechanisms have evolved that promote replication through DNA lesions. Increased error-prone bypass of DNA lesions causes increased mutagenesis, and as a consequence, a rise in the incidence of cancer. Error-free bypass processes, by contrast, keep mutagenesis low and reduce cancer frequencies. The goal of our project is to give insight into the mechanism and regulation of DNA damage bypass with special emphasis on the role of ubiquitin and SUMO modifications of DNA replication and repair proteins.

Genomic DNA can be damaged by both external environmental agents and endogenous metabolic processes. To maintain the integrity of the genome, a variety of repair mechanisms have evolved that remove damaged bases from DNA. However, as a result of mutations in repair systems, limited cellular repair capacity or timing, DNA damage sometimes is not repaired before replication takes place. When the DNA replication machinery encounters an unrepaired DNA lesion in the template strand, it faces a challenge, because the machinery is often unable to replicate past the lesion. Our research group is interested in the mechanisms that come into play when replication stalls at DNA lesions and that eventually lead to error-free or error-prone replication of damaged DNA. Error-prone replication of damaged DNA increases mutagenesis and leads to carcinogenesis, whereas error-free replication contributes to genetic stability.

The RAD6-RAD18-Dependent Pathway of Replication of Damaged DNA

Pathways for replication of damaged DNA are highly conserved from yeast to human cells. In the yeast Saccharomyces cerevisiae, the pathways are well characterized genetically, and both error-free and mutagenic modes of damage bypass require the yeast genes RAD6 and RAD18. Mutations in RAD6 and RAD18 confer a high degree of sensitivity to UV light and cause a defect in the replication of UV-damaged DNA. Furthermore, UV-induced mutagenesis does not occur in rad6 and rad18 mutants. Rad6-Rad18-mediated ubiquitin conjugation promotes replication through DNA lesions via three different pathways: the translesion synthesis pathways that depend on polymerase eta (Pol-eta) and Pol-zeta and the Rad5-dependent postreplication repair pathway.

Translesion Synthesis by Specialized DNA Polymerases

Pol-eta is unique among eukaryotic DNA polymerases in its proficiency at replicating through a UV-induced cis-syn thymine-thymine dimer. In humans, a defect in Pol-eta causes a variant form of xeroderma pigmentosum (XP-V). XP-V individuals are extremely sensitive to sunlight and they suffer from a high incidence of skin cancers. Pol-zeta promotes translesion synthesis by elongating the DNA strand from the nucleotides that were inserted opposite the damaged base by another DNA polymerase. By this mechanism, Pol-zeta promotes mutagenic translesion synthesis through certain DNA lesions.

RAD5-Dependent Error-Free Replication of Damaged DNA

The RAD5-dependent pathway, an alternative pathway to those that depend on translesion synthesis polymerases plays a major role in error-free replication of damaged DNA and in reducing UV-induced mutagenesis in yeast. Together with Mms2 and Ubc13, Rad5 forms a ubiquitin-conjugating enzyme complex that catalyzes the formation of polyubiquitin chains linked through the lysine-63 residue of ubiquitin. We have also shown that yeast Rad5 has DNA helicase activity that is specialized for replication fork regression. Rad5 action promotes a “copy choice” type of synthesis through DNA lesions, in which the newly synthesized daughter strand of the undamaged complementary sequence is used as the template for bypassing the lesion.

Human Homologues of Yeast Rad5: HLTF and SHPRH Tumor Suppressors

Recently, we have shown that HLTF and SHPRH are functional homologs of yeast Rad5. HLTF is frequently inactivated in colorectal and gastric cancers and SHPRH is frequently mutated in a wide variety of cancers. A requirement for HLTF and SHPRH in error-free postreplication repair of damaged DNA is in keeping with their cancer-suppression role.

Our present aim is to keep identifying new players in the human Rad6-Rad18 DNA damage bypass pathway which, similarly to hPoleta, SHPRH and HLTF tumor suppressors can be predicted to effect mutagenesis and carcinogenesis. We expect our research to provide greater insights into the areas of DNA repair, mutagenesis and carcinogenesis.