The VICC.ORG Investigator Directory

Ellen H. Fanning, Dr.rer.nat

Professor, Molecular Biology
Researcher

Contact Information:

Vanderbilt Biological Sciences Department
2325 Stevenson Ctr
Nashville, TN 37232
615-343-5677

Research Specialty:

DNA replication and damage repair in mammalian cells

Research Description:

Control of DNA replication is one of the key processes in the regulation of the mammalian cell cycle. Dysregulation of the cell cycle can arise at several stages, for example in the "decision" to begin DNA replication or in the proper completion of the S-phase, and is an important causative factor in a variety of human diseases, the most frequent being cancer. The long term goal of our research is to elucidate in molecular detail the mechanisms that control DNA replication in mammalian cells.

Replication of papovaviral DNA in infected cells and in cell-free reactions has proven to be an extremely useful model system toward this end. Synthesis of RNA primers is a key step in initiation of DNA replication. In the SV40 system, this step requires specific interactions among three proteins: the viral DNA helicase T antigen, and two cellular proteins, DNA polymerase-primase and replication protein A. We seek to map the protein interaction surfaces at the atomic level (cooperation with W. Chazin) and are using biochemical and genetic tools to elucidate how these interactions promote primer synthesis.

Another aspect of our research program focusses on the role of protein phosphorylation in regulating the initiation of DNA replication in a cell-free system. We have shown that phosphorylation of human DNA polymerase alpha-primase regulates its ability to synthesize RNA primers in a cell cycle-dependent manner. We are now testing the hypothesis that phosphate turnover on one of the polymerase-primase subunits responds to DNA damage/checkpoint signals.

DNA replication, repair, and recombination all require a DNA helicase activity to separate the two DNA strands and permit DNA processing. We have cloned a cDNA encoding a novel human DNA helicase that is required for the onset of chromosomal DNA replication. The function of the helicase is not known and orthologs have not been identified in lower eukaryotic genomes so far. The helicase appears in a focal pattern in the nucleus in G1, and most of it is exported to the cytoplasm in S phase. We have shown that the helicase interacts physically and functionally with DNA polymerase alpha-primase and replication protein A. Current work seeks to identify additional proteins that interact with the helicase and to elucidate its function using genetic and biochemical methods.

Mammalian chromosomes contain about 40,000 origins that direct initiation of chromosomal DNA at specific times in S phase. However, the cis-acting elements required to constitute a functional origin of DNA replication in mammalian chromosomes in vivo are not known. To address this question, we use a competitive PCR approach to assess initiation activity of a mammalian origin that has been stably integrated into ectopic sites in chromosomal DNA, as well as mutant versions of the origin. The ectopic origin is active in multiple chromosomal sites. Three essential sequence-specific elements in the origin have been identified so far. We have mapped the binding sites of initiation proteins in the functional origin and in mutant origins by chromatin immunoprecipitation. Future work will determine how initiation protein binding and chromatin structure cooperate to activate replication.

Publications:

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