NKTH Polányi Mihály Program (KFKT-1-2006-0010)
Title of the project: Role of novel tumor suppressor genes and modification of DNA repair proteins by ubiquitin in the replication of damaged DNA
Overall aims, objectives, and background of the research project
Cancer is one of the major causes of death in the present world. Although hundreds of research groups have been engaged with research on the 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 the idea that the roots of cancers lay in mutations of DNA, the inheriting 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 leads to the accumulation of damaged bases. Unrepaired DNA damages block the replication machinery, and stalled replication forks can lead to double strand DNA breaks and to major chromosomal rearrangements, ultimately to cell death. To avoid death, 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. However, cells have to pay a price for the survival: mutations accumulate in their DNA altering the genetic information and triggering carcinogenesis.
Postreplicative bypass processes come into play when the DNA replicational machinery encounters an unrepaired DNA lesion in the template strand and is unable to replicate past the lesion. Replication of damaged DNA templates can occur by translesion synthesis polymerases that are able to insert nucleotides opposite damaged bases, and by a yet unknown mechanism that may involve template switching to accomplish replication through the damaged site. The S. cerevisiae RAD6 and RAD18 genes are required for the error-free as well as mutagenic modes of damage bypass. Rad6, a ubiquitin-conjugating enzyme exists in vivo in a tight complex with Rad18, a DNA binding protein. Mutations in RAD6 and RAD18 confer a high degree of sensitivity to UV light, and they engender a defect in the replication of UV-damaged DNA. Also, UV-induced mutagenesis does not occur in either rad6D or rad18D mutants. The Rad6-Rad18-mediated ubiquitin conjugation promotes replication through DNA lesions via three different pathways: the DNA polymerase zeta, the DNA polymerase eta dependent translesion synthesis pathways and the Rad5-, Mms2-Ubc13-dependent postreplicational repair pathway.
In the last couple of years the research field of replication of damaged DNA has gone through a dramatic progress. Particularly, the discovery of special DNA polymerases, the so-called translesion synthesis polymerases radically changed our view of how cells can deal with DNA lesions that block the replicative DNA polymerases and how replication of damaged DNA can lead to carcinogenesis.
Ubiquitin-conjugation of PCNA stimulates damage bypass, while modification of PCNA by SUMO inhibits the Rad52-dependent recombination. Based on this finding, we proposed a mechanism, in which PCNA ubiquitylation leads to the displacement or disruption of the replication complex giving access to translesion synthesis polymerases and to the Rad5-dependent error-free alternative pathway of replication of damaged DNA. We have characterized in detail the interaction of poli with PCNA, and examined the effect of PCNA modifications on the interactions between PCNA and translesion synthesis polymerases. In the future, we plan to investigate PCNA ubiquitylation further, because it holds the key for the regulation of mutagenesis and carcinogenesis.
We have also initiated a new line of research on the Rad5 damage bypass pathway, of which preliminary results we have incorporated into our research proposal. As an alternative to translesion synthesis polymerases dependent pathways, the RAD5-dependent pathway plays a major role in the error-free replication of damaged DNA and in decreasing mutagenesis in yeast. Whereas the translesion synthesis polymerases dependent pathways have been characterized extensively, nothing is known about the molecular mechanism of the RAD5 pathway. Rad5 is a member of the SNF2/SWI2 family of proteins and exhibits a DNA dependent ATPase activity. It contains conserved helicase and ubiquitin ligase motifs, but no helicase or ubiquitin ligase activity has been shown for this protein. The mechanism of the Rad5 pathway is not known, but it is different from recombination, and it is hypothesized to operate through a “copy choice” type of mechanism, wherein the newly synthesized daughter strand of the undamaged complementary sequence is used as the template for bypassing the lesion.
The goal of our proposed project is to give more insight into the mechanism of mutagenesis and carcinogenesis. In particular, we plan to shed light on the role of yeast RAD5 gene and of its human homologues, which function as tumor suppressor, as well as on the ubiquitylation-dependent regulation of the replication of damaged DNA.
We will test our central hypothesis that yeast Rad5 protein has a replication fork specific DNA helicase activity as well as an ubiquitin ligase activity, and a Rad5-containing multisubunit enzyme complex carries out error-free replication through damaged DNA by template switching. We further hypothesize that yeast RAD5 has two functional homologues in humans, which are tumor suppressor genes and function in the replication of damaged DNA.
We have already identified two human genes as the closest human homologues of the yeast RAD5 gene. Human HLTF and SHPRH similarly to yeast RAD5 have a characteristic domain structure, in which the RING-finger domain is located in the middle of a helicase domain, and the both proteins share about thirty percent overall homology to yeast Rad5 protein. Importantly, very recently, each of these human gene has been shown to be tumor suppressors. In our proposed work, we will examine the possibility that human HLTF and SHPRH function in error-free replication of damaged DNA, which role could explain their tumor suppressor function.
Our proposed project will shed more light on the regulation and mechanism of DNA damage tolerance pathways. Our research on DNA lesion bypass has the potential to make a great impact on research on prevention of carcinogenesis and on the fight against cancer. Since many cancer drugs modify the bases of DNA, better understanding the mechanism and the regulation of replication through damaged DNA can open up new ways for the development of more effective cancer drugs. Therefore, in addition to basic research aspects, our expected results could give a strong ground for further applied science research.
Publications resulting from this project:
1. Chen, J., Ai, Y., Wang, J., Haracska, L. and Zhuang, Z. (accepted in 2009) Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis. Nat Chem Biol, 6, 270-272.
2. Unk, I., Hajdu, I., Blastyak, A. and Haracska, L. (accepted in 2009) Role of yeast Rad5 and its human orthologs, HLTF and SHPRH in DNA damage tolerance. DNA Repair (Amst), 9, 257-267.
3. Blastyak, A., Hajdu, I., Unk, I. and Haracska, L. (accepted in 2009) Role of double-stranded DNA translocase activity of human HLTF in replication of damaged DNA. Mol Cell Biol, 30, 684-693.
4. Jansen, J.G., Tsaalbi-Shtylik, A., Hendriks, G., Verspuy, J., Gali, H., Haracska, L. and de Wind, N. (2009) Mammalian polymerase zeta is essential for post-replication repair of UV-induced DNA lesions. DNA Repair (Amst), 8, 1444-1451.
5. Burkovics, P., Hajdu, I., Szukacsov, V., Unk, I. and Haracska, L. (2009) Role of PCNA-dependent stimulation of 3'-phosphodiesterase and 3'-5' exonuclease activities of human Ape2 in repair of oxidative DNA damage. Nucleic Acids Res, 37, 4247-4255.
6. Jansen, J.G., Tsaalbi-Shtylik, A., Hendriks, G., Gali, H., Hendel, A., Johansson, F., Erixon, K., Livneh, Z., Mullenders, L.H., Haracska, L. et al. (2009) Separate domains of Rev1 mediate two modes of DNA damage bypass in mammalian cells. Mol Cell Biol, 29, 3113-3123.
7. Acharya, N., Yoon, J.H., Gali, H., Unk, I., Haracska, L., Johnson, R.E., Hurwitz, J., Prakash, L. and Prakash, S. (2008) Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase eta in translesion DNA synthesis. Proc Natl Acad Sci U S A, 105, 17724-17729.
8. Zhuang, Z., Johnson, R.E., Haracska, L., Prakash, L., Prakash, S. and Benkovic, S.J. (2008) Regulation of polymerase exchange between Poleta and Poldelta by monoubiquitination of PCNA and the movement of DNA polymerase holoenzyme. Proc Natl Acad Sci U S A, 105, 5361-5366.
9. Unk, I., Hajdu, I., Fatyol, K., Hurwitz, J., Yoon, J.H., Prakash, L., Prakash, S. and Haracska, L. (2008) Human HLTF functions as a ubiquitin ligase for proliferating cell nuclear antigen polyubiquitination. Proc Natl Acad Sci U S A, 105, 3768-3773.
10. Blastyak, A., Pinter, L., Unk, I., Prakash, L., Prakash, S. and Haracska, L. (2007) Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression. Mol Cell, 28, 167-175.
11. Acharya, N., Haracska, L., Prakash, S. and Prakash, L. (2007) Complex formation of yeast Rev1 with DNA polymerase eta. Mol Cell Biol, 27, 8401-8408.
12. Acharya, N., Brahma, A., Haracska, L., Prakash, L. and Prakash, S. (2007) Mutations in the ubiquitin binding UBZ motif of DNA polymerase eta do not impair its function in translesion synthesis during replication. Mol Cell Biol, 27, 7266-7272.
