Pilot Projects
Project Title: A New Gene Involved in Genomic Stability and Cancer Prevention
Principal Investigator: Greg Freyer, Ph.D. (CEHNM)
Year: 2005
Award Amount: $25,000
Summary. In the human disease known as Bloom's syndrome, individuals suffer from a variety of ailments, including short stature, diminished intelligence and a very high risk of developing cancer. The cause of Bloom's syndrome is the loss of function of the gene BLM. By regulating the ability of cells to rearrange their DNA, Blm acts to maintain the integrity of the cell's genetic material. Blm is a DNA helicase and works with topoisomerase, Top3 in the role of genome caretaker. Studies by the Freyer laboratory in fission yeast, Schizosaccharomyces pombe, lead to the discovery of a new protein (Nce4/Rmi1) that appeared to act with the RecQ DNA helicase (Rqh1) and Topoisomerase III (Top3). This was based on findings that an nce4 mutant was lethal, but that this lethality was suppressed with the additional loss of Rqh1. A homologue of nce4 was also identified in budding yeast. This small and presumably essential protein poses great potential for identifying polymorphisms that lead to reduced or altered function. Such polymorphisms would presumably decrease the stability of the genome and increase the risk of cancer in humans. The goal is to study the role of Rmi1/Nce4 in genomic stability and to identify potential polymorphisms in human RMI1/NCE4 from immortalized cells and to determine if they are more prevalent in cancer patients. Such findings would provide a marker for identifying individuals for increased risk of developing cancer.
Results. Due to the fact that there were several clues as to how Rmi1 might function, some additional studies in fission yeast were initially carried out. Loss of the top3 gene, which codes for topoisomerase III, is lethal in S. pombe, unless rqh1+ is also deleted; a result similar to that found is nce4 mutants. In S. cerevisiae, a Sgs1-Top3 fusion protein was made that functioned like wild type cells. Finally, bacterial topoisomerase I could complement loss of TOP3. The role of Rmi1 was speculated to bridge Rqh1 with Top3. To test this hypothesis, using a strategy implemented in S. cerevisiae, a chimeric construct was created to fuse Rqh1 with Top3. This was then crossed with a nce4-delta rqh1-delta mutant strain, and looked for recovery of a nce4-delta rqh1-top3 fusion strain. A screen of multiple strains failed to find any of the desired clones. While a negative result, these data argue against Rmi1 as simply acting to link Rqh1 and Top3.
The next goal of this Pilot Project was to create a human cell line that was deficient in hRMI1. To accomplish this, hRMI1 was knocked-down using RNA interference technology. A region of 60 base-pairs containing the sequence complementary to the 5' region of hRMI1 were cloned into the pSUPERIOR vector. This vector was to be cloned into the human 293T cell line. When this system was on the verge of testing, a paper was published on the human homologue of RMI1, which they named BLAP-75, showing that it was part of the human BLM-Top3 complex (Yin et al., EMBO J., 2005, 24:1465 2005). This publication preceded three other papers from the Yin and two other laboratories on human BLAP75 that largely accomplished the aims of this pilot (with the exception of the third aim) to screen human lymphocytes for possible polymorphisms in hNCE4. This work is ongoing.
Outcomes/Publications.
Chang M, Bellaoui M, Zhang C, Desai R, Morozov P, Delgado-Cruzata L, Rothstein R, Freyer GA, Boone C, Brown GW. RMI1/NCE4, a suppressor of genome instability, encodes a member of the RecQ helicase/Topo III complex. 2005. EMBO J. 24:2024-33.
Grants Submitted:
One R01 grant (CA116354), "A New Member of the RecQ-Top3 Genome Stability Complex", was submitted and is currently being revised for resubmission.