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Regulation and mechanism of replication of damaged DNA: role of yeast and human Rad5 and ubiquitylation

Final Activity Report Summary - DNA DAMAGE BYPASS (Regulation and mechanism of replication of damaged DNA: role of yeast and human Rad5 and ubiquitylation)

Cancer is one of the major causes of death in the present world. Although hundreds of research groups have been engaged in the research of this disease, 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 that the roots of cancers lay in mutations in DNA, the inheriting material of cells. DNA damages, caused by extrinsic or intrinsic agents, such as ultraviolet radiation or free reactive oxygen species are usually removed from DNA and repaired by one of the several DNA repair systems of the cell, which preserves the genetic information. However, high exposure to DNA damaging agents can lead to the accumulation of damaged bases. Unrepaired DNA damages block the replication machinery, which can lead to chromosomal rearrangements or 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 yeasts, at least three alternative pathways of replication of damaged DNA operate: the polymerase zeta dependent error-prone, the DNA polymerase eta dependent error-free, and the Rad5-dependent error-free post replication repair pathway, which are conserved 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 humans, a defect in polymerase eta causes the variant form of Xeroderma pigmentosum (XP-V). Consistent with the role of Poleta in the error-free bypass of UV lesions, XP-V cells are hypermutable with UV light and as a consequence, XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. While the two DNA polymerase dependent pathways are well characterised, our knowledge about the Rad5 dependent error-free pathway has been in a rudimentary stage. It was not known how the Rad5-dependent pathway stimulates error-free replication of damaged DNA in yeasts, and whether a similar pathway in humans operates.

The aim was to exploit the role of Rad5 in error-free replication of damaged DNA in yeast and human cells by employing advanced tools of yeast genetics, molecular biology, biochemistry, and enzymology. Our research group have found that Rad5 has a replication fork-specific helicase activity that can promote a copy choice type of DNA synthesis, in which the damage is bypassed by template switching using the newly synthesised DNA strand as the template that is formed by replication fork regression. Therefore, while translation synthesis polymerases copy DNA directly from the damaged template, the Rad5-dependent post replication repair operates via using the information of the undamaged newly synthesised nascent strand on the sister duplex. We have also identified SHPRH as a human homologue of yeast Rad5. A tumour suppressor function for SHPRH is indicated by that the SHPRH gene is mutated in a number of melanoma and ovarian cancer cell lines.

Our finding that SHPRH has a role in DNA damage bypass indicate that by preventing mutagenesis, SHPRH DNA repair function would contribute to minimising the incidence of carcinogenesis in humans. In summary, our research on yeast Rad5 and on human SHPRH has contributed to a better understanding of the molecular mechanism of mutation avoidance and carcinogenesis.