DNA Replication, Repair, and Apoptosis Nucleases in Genome Stability and Cancer
DNA replication and repair are critical for maintaining genome stability. These processes are in part dependent on the activities of an emerging family of structure-specific nucleases. Flap EndoNuclease 1 (FEN1) is a metallo- and substrate structure specific- nuclease. It possesses three distinct biochemical activities, functioning as a flap endonuclease (FEN), a nick-specific exonuclease (EXO), and a gapdependent endonuclease (GEN). FEN1 plays a critical role in maintaining human genome stability via six different pathways. It serves as a major nuclease for RNA primer removal during Okazaki fragment maturation and for long patch base excision repair using its FEN activity. Its concerted action of EXO and GEN activities is critical in resolution of di- and tri- nucleotide repeat secondary structures and stalled DNA replication forks, as well as in apoptotic cell DNA fragmentation. It also plays a major role in maintenance of telomere stability.
The multiple functions of FEN1 are regulated via three major mechanisms: formation of complexes with different protein partners, cellular compartmentation, and post-translational modifications. More than 30 proteins have been identified to interact with FEN1, forming specific complexes in different pathways. Upon acetylation, FEN1 translocates into the nucleus in response to DNA damage and cell cycle phase changes. It is very much enhanced in the nucleolus for maintenance of stability of tandem repeats of ribosomal DNA. FEN1 is also in mitochondrion, playing an important role in mitochondrial DNA replication and repair. The nuclease is acetylated, phosphorylated or methylated in different molecular events and the interaction between methylation and phosphorylation determines its recruitment onto DNA replication forks via proliferating cell nuclear antigen. The first group of FEN1
somatic mutations has been identified in human cancer cells, which has clear segregation of biochemical activities. The future emphasis will be placed on the mutations and prevalent polymorphisms that may impair one of the three major regulatory mechanisms. See Project 1
- Functional Analysis of FEN-1 Nuclease in Genome Stability.
Recently, we found that another major nuclease, DNA2, is dominantly localized into mitochondria and cooperatively processes replication and repair DNA intermediates for ligation and completion of circular mtDNA replication and repair. These novel and exciting observations prompted us to: i) knock out the DNA2 gene in mice to determine if defective DNA2-mediated RNA primer removal causes mitochondrial genomic instabilities, consequently promoting cancers and other genetic diseases, and ii) link functional defects of the DNA2 mutations identified in human mitochondrion-based diseases to pathologic mechanisms. Information made available from these studies should establish a relationship among the functions of these novel mitochondrial genes, unique mitochondrial mutagenic phenotype(s), and pathological mechanisms. The proposed study may also establish a foundation for the development of new treatment regimens for patients with mitochondrion-based cancers and other disorders. See Project 2
- Role of Nucleases in RNA Primer Removal and Mutagenesis.
The other novel nuclease that we are interested in is called TatD, which possesses a nick and 3’ exonuclease activity and is involved in apoptosis DNA fragmentation. In collaboration with Dr. John Williams in the Department of Molecular Medicine, we are currently undertaking a detailed 3-D structural and functional analysis of TatD to determine its role in apoptosis and the biological consequences, in human cells, of defects in this nuclease.