|Institution||College of Medicine|
|Address||500 University Drive Hershey PA 17033|
Professor of Pathology, and Biochemistry and Molecular Biology
GRADUATE PROGRAM AFFILIATIONS:
Biochemistry and Molecular Biology, Cell and Molecular Biology, Genetics, MD/PhD Degree Program, Integrative Biosciences
Ph.D., University of Wisconsin-Madison, 1988
Postdoctoral Training, NIH/National Institute of Environmental Health Sciences, 1988-1993
The process of mutation is a cornerstone of Genetics. My laboratory’s research goal is to make significant scientific contributions towards the understanding of mutational processes in human cells, particularly in the context of genome evolution and disease. The process of mutagenesis represents a double-edged biological sword. On one hand, mutations are essential for life, and provide the necessary fuel driving evolution. One the other hand, uncontrolled mutations result in genome instability and disease. Our specific research niche is elucidating the role of repetitive DNA sequences in genome stability. Over 50% of the human genome is encoded by repetitive DNA elements of various types: satellite, minisatellite and microsatellite repeats. Despite their prevalence and postulated biologic functions, very little is known regarding the stability of repetitive elements in the human genome and the mechanisms of DNA replication and repair ensuring stability.
A major focus of my laboratory is on microsatellite sequences, short tandem repeats of 1-6 units per motif. Microsatellites are present on every chromosome and are highly polymorphic in human populations. Microsatellites can be important regulators of gene expression, affecting transcription rate, RNA stability, splicing efficiency, and RNA-protein interactions. Consequently, allele-length polymorphisms at common microsatellites are implicated as genetic risk factors in several diseases. The full impact of microsatellite changes on genome function has yet to be elucidated; therefore, it is of utmost importance to gain knowledge about how microsatellites arise, mutate, and eventually cease to exist at individual loci in the human genome. The goal of our interdisciplinary project, funded by the NIH, is to elucidate mechanisms defining the microsatellite life cycle in the human genome. This team project represents a collaboration integrating experimental and computational approaches. Genome-wide trends of microsatellite evolution are uncovered computationally (through analysis of sequenced primate and individual human genomes) in the Makova laboratory (PSU-UP campus), and are used to formulate hypotheses. Concomitantly, specific mechanisms are tested experimentally using the Eckert laboratory’s in vitro and ex vivo mutagenesis assays. The results of this project promise to be of considerable significance for understanding the dynamics of human genome evolution. Additionally, our research may have direct relevance to the issues of public health and clinical genetics. For example, the new information gained by our research can be used to predict the probability of each microsatellite to undergo mutation or cease to exist, and the probability of any genomic region to bear a new microsatellite. This will have major importance for assessing an individual’s disease risks, especially in the era when individual human genomes are being rapidly sequenced.
Our laboratory also is examining the role of microsatellite sequences in tumor cell genome evolution. Our working hypothesis is that the loss of biochemical pathways regulating microsatellite stability contributes to tumor genome evolution by producing phenotypic variants in key cancer-associated loci. Colorectal carcinomas (CRC) provide us with a relevant framework in which to examine our hypothesis. Microsatellite instability (MSI) is observed in numerous cancer types, but the underlying mechanistic basis is known for only a subset of tumors: those due to loss of mismatch repair (MMR). We have demonstrated that intrinsic DNA features (motif size, length, and sequence composition) contribute significantly to mutagenesis of common microsatellites, and that each motif differs with regard to mutational dynamics. In addition to MMR, our experiments have revealed that other mechanisms likely exist in human cells to ensure microsatellite stability. We hypothesize that complete and accurate human genome replication, particularly of repetitive DNA elements, requires the coordinated activities of multiple specialized DNA polymerases.
A second focus of my laboratory is replication-based mechanisms underlying structural variation at common fragile sites. Common fragile sites (CFS) are regions of the genome prone to chromosomal instability in tumor cells, but the precise DNA sequence features that characterize this fragility are not known. The most widely accepted model for chromosome breakage within CFS posits that DNA replication fork stalling precedes DNA breakage and subsequent DNA rearrangements. My laboratory has discovered that several repeated DNA elements, including microsatellites and inverted repeats, are inhibitory to replicative DNA polymerases in vitro, and that specialized DNA polymerases/ replication proteins are required to complete replication through such repeated sequences. Many microsatellite sequences have the potential for adopting non-B form DNA conformations, including Z-DNA, H-DNA (triplex) and cruciform structures. We are striving to elucidate the role that DNA secondary structure plays in human cell mutagenesis and genome evolution. We hypothesize that DNA replication inhibition within CFS is due to the density and/or arrangement of specific repetitive sequences, relative to other areas of the genome. To test this hypothesis, we are using a combination of biochemical, genetic and computational approaches. The biochemical and genetic experiments will elucidate DNA replication dynamics through CFS and the mechanisms/endogenous factors responsible for DNA breakage in human cells. Computationally, Dr. Makova’s lab is develop an algorithm to predict novel chromosomal sites at risk for instability. The identification of key cis-acting elements and trans-acting proteins may lead to understanding individual genetic risk factors and environmental exposures that act to increase chromosomal instability during neoplastic progression.
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