"Scientists see what anybody else, but they think of ideas that nobody has thought before"

 

Albert Szent-Györgyi

Replication of Damaged DNA in Yeast and Human Cells: Implications for Mutagenesis and Carcinogenesis

 

Genomic DNA is subjected to damage 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, sometimes DNA damage is not repaired before replication takes place due to mutations in repair systems, limited cellular repair capacity, or timing. When the DNA replication machinery encounters an unrepaired DNA lesion in the template strand, it faces challenging problems 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 finally lead to either error-free or error-prone replication of damaged DNA. In humans, error-prone replication of damaged DNA causes increased mutagenesis and leads to carcinogenesis, whereas error-free replication contributes to genetic stability. We expect our research to provide greater insights into areas of DNA repair, mutagenesis, and carcinogenesis.

 

The RAD6-RAD18-dependent pathway of replication of damaged DNA The pathways for replication of damaged DNA are highly conserved from yeast to human cells and are well characterized genetically in the yeast Saccharomyces cerevisiae. The yeast RAD6 and RAD18 genes are required for 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 engender a defect in the replication of UV-damaged DNA. Furthermore, UV-induced mutagenesis occurs in neither rad6 nor rad18 mutants. Rad6-Rad18-mediated ubiquitin conjugation promotes replication through DNA lesions via three different pathways: the polymerase (Pol) eta·- and Pol kappa-dependent translesion synthesis pathways 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; genetic studies in yeast as well as in human cells have indicated a role for Pol eta· in promoting error-free replication through this damage. In humans, a defect in Pol eta· causes a variant form of xeroderma pigmentosum (XP-V). Consistent with the role of Pol eta· in the error-free bypass of UV lesions, XP-V cells are hypermutable with UV light and, as a consequence, XP-V individuals have a high incidence of sunlight-induced skin cancers. Our experiments have indicated that Pol eta· is also able to replicate through many other DNA lesions. For example, yeast Pol eta· replicates efficiently and accurately through an 8-oxoguanine lesion, formed by the attack of oxygen-free radicals on bases in DNA; our genetic studies in yeast verified a role for Pol eta· in minimizing the incidence of mutations arising from misincorporation errors opposite this lesion. In addition, We have shown that yeast Pol kappa promotes translesion synthesis by extending from the nucleotides inserted opposite the damaged base by another DNA polymerase. By this mechanism, Pol kappa promotes mutagenic translesion synthesis through certain DNA lesions such as UV-induced lesions and abasic sites. We have also characterized human Pol iota and Pol zeta, orthologs of Pol eta·. These translesion synthesis polymerases exhibit low fidelity on undamaged DNA, with Pol iota even violating the Watson-Crick base-pairing rule. Pol iota can insert nucleotides opposite many DNA lesions; we have shown that Pol iota and Pol kappa act in concert to bypass certain DNA lesions. Pol zeta, however, resembles Pol kappa in inefficiently inserting nucleotides opposite DNA lesions, but it is efficient at extending from bases opposite certain DNA lesions.

Now, we are investigating how the translesion synthesis polymerases get recruited to the stalled replication fork and what determines which translesion polymerase gains access to the damaged bases.

 

The RAD5-dependent error-free replication of damaged DNA As an alternative to pathways dependent on translesion synthesis polymerases, the RAD5-dependent pathway plays a major role in error-free replication of damaged DNA and in decreasing UV-induced mutagenesis in yeast. Whereas the pathways dependent on translesion synthesis polymerases have been characterized extensively, much less is known about the molecular mechanism of the RAD5 pathway. Recently, we have shown that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression, as Rad5 can concertedly unwind and anneal the nascent and the parental strands of the fork without exposing any single-stranded regions. This Rad5 activity would ensure damage bypass by promoting replication fork regression where the newly synthesized DNA strand of the sister duplex can be used as a template. In addition to its role as a DNA helicase, Rad5 has a C₃HC₄ RING domain that is indicative of a ubiquitin ligase function. In DNA-damaged yeast cells, Rad5 is required together with the Mms2-Ubc13 ubiquitin-conjugating complex for the polyubiquitination of PCNA at its lysine 164 residue via a lysine 63-linked ubiquitin chain. In vivo, Rad5 associates with the Mms2-Ubc13 complex via Ubc13, and it also interacts with the Rad18 subunit of the Rad6-Rad18 complex, and presumably through these interactions, coordinates the action of these two enzyme complexes in PCNA polyubiquitination. Importantly, the inactivation of the DNA helicase function or the ubiquitin ligase function of Rad5 causes the same high degree of PRR defect as is seen in the rad5 delta mutant, indicating that both these activities are indispensable for Rad5 function in PRR.
Our goal is to elucidate the mechanism of the yeast RAD5 pathway by examining the enzymatic activity of Rad5 protein and by using an it vitro reconstituted damage bypass system. We are also addressing the question whether a homologue yeast Rad5-like damage-bypass system operates in human cells, as well.

 

Role of proliferating cell nuclear antigen (PCNA) in DNA damage bypass PCNA is an integral part of the replication complex, where it binds to the replicative polymerases Pol eta´ and Pol etaµ and keeps them in close proximity to the primer-template junction. We have shown that several translesion synthesis polymerases, such as Pol eta·, Pol iota, and Pol zeta, can also interact with PCNA, which stimulates the DNA synthetic activity of these polymerases. Furthermore, our genetic data have demonstrated that PCNA binding is essential for the in vivo function of yeast Pol eta·. Because of its role in replication and its ability to bind to replicative and translesion synthesis polymerases, PCNA could be the molecular link that tethers translesion synthesis polymerases to the replication fork and could mediate polymerase exchange at DNA lesion sites.

Upon DNA damage, PCNA becomes monoubiquitinated by Rad6-Rad18 and can then become polyubiquitinated in a Rad5- and Mms2-Ubc13-dependent manner. In addition, during the S phase of the cell cycle, PCNA can be modified by SUMO. Recently, we showed that, while ubiquitin-conjugation to PCNA facilitates damage bypass, modification of PCNA by SUMO inhibits Rad52-dependent recombination. On the basis of this study, we have proposed a mechanism in which PCNA ubiquitylation leads to displacement or disruption of the DNA replication complex, giving access to translesion synthesis polymerases and to the Rad5-dependent error-free alternative pathway of replication of damaged DNA.

One of our goals is to understand the role of PCNA modifications further, particularly in the switch from the replicative polymerase to a translesion synthesis polymerase and in decision making between error-free and error-prone pathways of replication of damaged DNA.

Function of human SHPRH ubiquitin-ligase in damage bypass Human SHPRH gene is located at the 6q24 chromosomal region, and loss of heterozygosity in this region is seen in a wide variety of cancers. SHPRH is a member of the SWI/SNF2 family of ATPases/helicases, and it possesses a RING motif characteristic of ubiquitin ligase proteins. In both of these features, SHPRH resembles the yeast Rad5 protein. Recently, we have shown that SHPRH is a functional homolog of Rad5. Similar to Rad5, SHPRH physically interacts with the Rad6-Rad18 and Mms2-Ubc13 complexes, and we found that SHPRH protein is an ubiquitin ligase indispensable for Mms2-Ubc13-dependent polyubiquitylation of proliferating cell nuclear antigen. Based on these observations, we predict a role for SHPRH in promoting error-free replication through DNA lesions. Such a role for SHPRH is consistent with the observation that this gene is mutated in a number of cancer cell lines, including those from melanomas and ovarian cancers, which raise the strong possibility that SHPRH function is an important deterrent to mutagenesis and carcinogenesis in humans. We are interested in exploring further the enzymatic activity of SHPRH and its role in postreplication repair.