Assistant Professor
Ph.D., Stanford University, 1994
American Cancer Society Postdoctoral Fellow, Cold Spring Harbor, 1995-2003

Program Affiliation: Molecular Biology & Genetics

Research Groups Affiliation: Biochemistry

Phone: 812/855-8514
Fax: 812/855-6082
Email Viola

DNA replication requires the concerted effort of over 30 proteins that assemble at specific sites on the chromosome referred to as origins of DNA replication. Once assembled, they form a "machine" that coordinates the simultaneous synthesis of copies of both strands of the chromosome at an amazing speed with incredible accuracy.   The task is arduous, for not only must DNA synthesis on both strands remain coupled to minimize the introduction of alterations in the DNA sequence, but the replication machine must also navigate effectively protein roadblocks that function to regulate gene expression.   To add to the complexity, the replication machine must also interface with two other processes that occur concomitantly with DNA replication- the assembly of newly synthesized DNA helices into chromatin, and the physical linking of the DNA helix replicas or sister chromatids until their partitioning later in the cell cycle.   Although infrequent, problems with DNA replication do occur and, if not repaired, can result in potentially deleterious alterations (mutations) in chromosomes.   Consequently, the cell harbors DNA surveillance pathways (DNA repair and cell cycle checkpoint pathways) that prevent inheritance of altered chromosomes by halting the cell division cycle to allow chromosomes to be fixed.   In the absence of DNA surveillance pathways, alterations occur at a higher frequency--a state called genomic instability--leading to the accumulation of mutations that can result in human disease.   

My interests are:

1. how the human replication machine assembles and maintains its integrity while replicating the genome
2. how the replication machine interfaces with the establishment of sister chromatid cohesion (process of linking of sister chromatids)
3. how are architectural problems with the replication machine and, consequently, mistakes made by a defective replication machine monitored and fixed.

To study the human DNA replication machine, we use an in vitro model viral DNA replication system that employs a viral chromosome and one viral protein.   The viral protein functions to bind the viral origin of DNA replication and recruit components of the human replication machine.   Once recruited, they assemble into a replication machine that replicates the viral chromosome as if it was a human chromosome. Therefore we can analyze human DNA replication machine function on a defined DNA substrate using protein biochemistry (Mass Spectrometry, recombinant protein expression etc.) and molecular biology techniques.   Using this replication system, proteins identified as essential for human replication machine assembly and function include the group of proteins we are currently studying, the Replication Factor C, or RFC, protein family.   To date, this family consists of 8 structurally related proteins, four small (RFC2, RFC3, RFC4, and RFC5) and four large (RFC1, RAD17, CTF18, and ELG1), of very similar DNA and amino acid sequence.   They form four distinct complexes composed of all four small proteins with only one large protein (see Figure 1). Surprisingly, although these complexes are very similar in primary structure and function, their activities are not redundant, but essential in four distinct pathways required for chromosome duplication, inheritance, and maintenance.    The founding family member RFC (defined by RFC1) is essential for replication machine assembly and function, whereas another family member, CTF18, is required for the establishment of sister chromatid cohesion.   The other two family members, RAD17 and ELG1, function in DNA surveillance pathways that monitor for and facilitate repair of any alterations in the genome.

The fundamental function of the RFC complex family is to assemble or "load" other protein complexes onto DNA, called clamps, which are involved in DNA replication, recombination, and DNA repair (see Figure 2). We are interested in not only the mechanism of clamp loading by the RFC family, but also, given their structural and functional similarity, how RFC family members are targeted to and work in distinct pathways.   Our efforts are devoted to (1) defining the determinants for pathway specificity; (2) understanding how the cell regulates assembly of all the RFC family complexes given they share a common sub-complex or "core" of the four small proteins; and (3) elucidating the mechanism of clamp loading and clamp function.   Many of the proteins that function in DNA surveillance pathways, such as RFC protein family are either essential for life, or loss of their function is associated with various forms of human disease, such as cancer, developmental and neurological disorders. One long term goal is to understand at a molecular level how genomic instability promoted by loss of function of RFC protein complexes contributes to perturbations in cell and hence organismal development.

Ellison, V. and Stillman. (2003) Biochemical Characterization of DNA Damage Checkpoint Complexes: Clamp Loader and Clamp Complexes with Specificity for 5' Recessed DNA. PloS , 1 (2 ) , 1-13.  

Ellison, V. and Stillman, B. (2001) Opening of the clamp: an intimate view of an ATP-driven biological machine. Cell , 106 , 655-660.

Ola, A., Waga, S., Ellison, V., Stillman, B., McGurk, M., Leigh, I.M., Waseem, N.H. and Waseem, A. (2001) Human-Saccharomyces cerevisiae proliferating cell nuclear antigen hybrids: oligomeric structure and functional characterization using in vitro DNA replication. J Biol Chem , 276 , 10168-10177.

Ellison, V. and Stillman, B. (1998) Reconstitution of recombinant human replication factor C (RFC) and identification of an RFC subcomplex possessing DNA-dependent ATPase activity. J Biol Chem , 273 , 5979-5987.