Marila Cordeiro–Stone, PhD
My research is focused on mechanisms of DNA replication, DNA repair, and cell cycle checkpoints in human cells. Inherited and acquired defects in the molecular network of protection of genetic stability are associated with increased risk for mutations underlying cancer pathogenesis. My laboratory studies how human cells succeed in completing genome duplication, even in the presence of DNA lesions that block the replication machinery. DNA damage tolerance depends on the replication fork protection complex avoiding the collapse of stalled forks, the recruitment of specialized DNA polymerases for catalysis of translesion synthesis (TLS), and the return of the nascent DNA back to the high fidelity, replicative DNA polymerases. In addition to TLS, recombination-based, post-replication repair pathways might also contribute to eliminating or minimizing the formation of daughter strand gaps opposite template lesions (potential precursors to DNA double strand breaks). DNA polymerase eta (pol eta) is capable of replicating past cyclobutane pyrimidine dimers (CPD), the major DNA photoproduct induced by ultraviolet (UV) radiation. The importance of human pol eta in reducing mutagenesis and decreasing risk for skin cancers associated with sun exposure is illustrated by the xeroderma pigmentosum variant (XP-V) syndrome. XP-V patients lack pol eta expression and are over 1,000 times more susceptible than are normal individuals to skin cancers, including malignant melanoma.
My laboratory used XP-V cells to investigate the underlying defect responsible for their enhanced sensitivity to UV-induced mutagenesis and to search for the identity of the XP-V gene product. Cell biology studies and development of biochemical assays with specificity and sensitivity to detect translesion synthesis of a single CPD in double stranded DNA helped to build the foundation for the discovery of DNA pol eta by other laboratories. Initial studies were expanded to include analyses of the structure of DNA replication intermediates, the evaluation of translesion synthesis versus template switching (copy choice) as the primary mode of replication past thymine dimers in vitro, and the effect of the position of the DNA lesion (either on the template for the leading or the lagging strand of nascent DNA) on the efficiency of bypass of DNA photoproducts.
Complementation of expression of wild-type pol eta in XP-V cells generated isogenic pairs of diploid human fibroblasts for studies of the biological role(s) of this specialized polymerase in DNA damage tolerance, following exposure to either physical or chemical carcinogens. These isogenic cells represent valuable tools for studies of mechanisms for recruiting pol eta to replicate across DNA lesions and keeping it from replicating undamaged DNA, given that the replication fidelity of pol eta is significantly lower than that exhibited by the normal replicative DNA polymerases. A study in collaboration with the Chaney laboratory indicated that pol eta is also involved in accurate translesion synthesis of certain DNA adducts formed by cisplatin, a drug commonly used in cancer chemotherapy.
DNA damage checkpoints cooperate with nucleotide excision repair to minimize the risk associated with replicating DNA containing lesions by inhibiting entry of cells into the S phase of the cell cycle or delaying initiation at banks of replicons not yet activated. My laboratory is particularly interested in the signaling pathways that underlie the intra-S phase checkpoint responses of inhibition of replicon initiation and rate of replication fork displacement in human cells exposed to UV. In collaboration with the W. Kaufmann laboratory, we demonstrated the dependence of the S checkpoint inhibition of replicon initiation on the activity of the ATR and Chk1 kinases in human cells exposed to low fluences of UV and the importance of cell cycle checkpoints in protecting XP-V fibroblasts from UV-induced chromatid-type aberrations. Ongoing studies are focused on the role of Timeless, Tipin, and other components of the replication fork protection complex in regulating DNA replication in both control and UV-irradiated human cells.
Understanding the regulation and organization of DNA replication requires information on the chromosomal location and time of activation of replication origins during the S phase of the cell cycle. Our collaborative studies with the D. Kaufman group have led to the generation of a cosmid library of DNA sequences replicated early in the S phase of diploid human fibroblasts and the discovery of several new origins of replication. Immuno-staining of individual DNA fibers spread on glass slides has led to new studies of the dynamics of DNA replication and intra-S checkpoint responses to DNA damage in human cells.
Current efforts are directed toward the identification of key molecular events that contribute to the development of melanoma in skin areas exposed intermittently to sunlight. The component of solar radiation that is most damaging to the skin is represented by the shortest UV wavelengths that penetrate the Earth atmosphere (UVB). Therefore, it is our goal to translate past research experience with biological effects of UV in cultured fibroblasts to a more clinically relevant cell type. In this regard, my laboratory is collaborating with a group of basic scientists and clinical investigators with expertise in different aspects of DNA repair, replication, and checkpoint control in UV-damaged cells, as well as pathogenesis of malignant melanoma. Such a multidisciplinary effort is needed for reaching a better understanding of how several DNA metabolic pathways and cellular responses to DNA damage are coordinated in the target cell for malignant melanoma (e.g., human melanocytes) and how these protective barriers might be compromised during cancer development. Correlations of clinical behavior of melanomas with molecular genetics and patterns of gene expression will inform the establishment of new classification systems and choice of individualized approaches for treatment of a disease that is increasing in incidence and for which there is great need for effective therapy.
View list of publications from PubMed