EXCEED THE SPACE PROVIDED. Prokaryotes and eukaryotes are endowed with multiple specialized DMApolymerases that support DNA synthesis across sites of DNA damage that arrest normal high-fidelity replication. This process is called translesion DNA synthesis (TLS). This function is fundamental to the survival of cells that have suffered arrested DNA replication due to the presence of unrepaired DNA damage, and to the genesis of mutations in living cells. These polymerases may also play a role in somatic hypermutation (SH) in the immune system by filling in gaps in DNA in an error-prone manner, increasing the diversity of antibodies. Understanding the molecular mechanism of TLS is therefore central to understanding all human diseases associated with mutagenesis, especially cancer, and to understanding SH in the immune system. We have identified a polymerase, POLK, and created a mouse model for this damage inducible polymerase. The progeny of Polk- /- mice manifest a mutator phenotype and cells from these mice are sensitive to killing by UV radiation and to the polycyclic aromatic mutagen and carcinogen benzo[a]pyrene.
Specific aim 1 of this renewal proposal is to characterize the molecular basis of the mutator phenotype identified in progeny of Polk-/- mice by using mutation detection systems to clarify the in vivo functions of POLK. These systems will identify changes found in the DNA of mice lacking this enzyme. Additionally, we have discovered multiple mRNA isoforms of POLK in the testis of these mice and preliminary evidence indicates that some of these are translated to proteins.
Aim 2 is to identify the functions of the multiple POLK isoforms present in mouse testis, their biochemical interactions with other proteins and DNA as well as their ability to add nucleotide opposite DNA lesions. We have also identified proteins that interact with POLK and are pursuing how these interactions affect damage recognition and repair.
Aim 3 is to clarify the molecular mechanism(s) of polymerase switching from replicative to TLS polymerase, in particular the role of REV1L protein (a protein that interacts with several polymerases) in this process. The results of these studies will clarify the in vivo functions of POLK and lead to further understanding of how POLK and it's protein isoforms interact with REV1L during polymerase switching will help define how this aspect of genome maintenance works to prevent mutations that lead to cancer and other diseases. PERFORMANCE SITE ========================================Section End===========================================
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| Guo, Caixia; Sonoda, Eiichiro; Tang, Tie-Shan et al. (2006) REV1 protein interacts with PCNA: significance of the REV1 BRCT domain in vitro and in vivo. Mol Cell 23:265-71 |
| Guo, Caixia; Tang, Tie-Shan; Bienko, Marzena et al. (2006) Ubiquitin-binding motifs in REV1 protein are required for its role in the tolerance of DNA damage. Mol Cell Biol 26:8892-900 |
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