MURIEL W. LAMBERT
Northwestern University, 1970
responses to DNA damage
The DNA repair defect in Fanconi anemia
Damage to DNA is an important etiologic pathway for a number of important processes such as cancer, birth defects and aging. The research programs in my laboratory focus on mechanisms of repair of damage that occurs to cellular DNA in mammalian cells. Of primary importance are investigations of these repair processes in genetic diseases in which there are known DNA repair defects. One of these diseases is the cancer prone, hematological disorder, Fanconi anemia (FA). Patients with this disorder suffer from bone marrow failure that leads to aplastic anemia and leukemia. These patients have a DNA repair defect, specifically in repair of DNA interstrand cross-links. We are carrying out research to determine the proteins involved in this repair defect and in relating this defect to the clinical manifestations of the disorder. We have recently shown that cells from six of the FA complementation groups tested are deficient in the structural protein, nonerythroid a spectrin ( a SpII S *). These studies demonstrate that a SpII S * forms a complex in the nucleus with the FANCA, FANCC, FANCF and FANCG proteins and with XPF, a protein involved in repair of DNA interstrand cross-links. In normal cells, these proteins co-localize to discrete nuclear foci after the cells are damaged with a DNA interstrand cross-linking agent. In FA cells, these nuclear foci are greatly decreased. We have proposed a model in which a SpII S * acts as a scaffold to help recruit and align the FA and DNA repair proteins at sites of damage and aid in their role in the repair process. In FA cells, where there are reduced levels of a SpII S *, there is decreased recruitment of proteins to sites of DNA damage and decreased DNA repair.The nature of these protein interactions and their importance in the repair process is currently being investigated. Since a spectrin is involved in a number of different cellular processes, a deficiency in this protein could have far reaching consequences and could thus possibly account for some of the diverse cellular and clinical defects that have been observed in FA cells.
Role of chromatin structure in DNA repair
Chromatin structure plays a critical role in modulation of cellular response to DNA damage. We are examining how it influences the ability of proteins involved in the repair process to interact with damaged DNA. The cancer prone, DNA repair deficient genetic disorder, xeroderma pigmentosum (XP), is being used as a model for these studies. Cells from XP complementation group A (XPA) patients, unlike normal human cells are defective in ability to repair damaged DNA when it is in the form of nucleosomes. Studies in my laboratory suggest that this is related to the mechanism by which DNA endonucleases involved in the repair process locate sites of damage. These studies indicate that the XPA protein may be involved in determining the mechanism of action utilized by these endonucleases. By investigating the role that the XPA protein plays in location of damage sites on DNA and the importance of this protein for location of target sites on nucleosomal DNA, we expect to obtain a clearer understanding of the mechanism of interaction of repair proteins with damaged nucleosomal DNA and the severe consequences, such as the production of numerous cancers, that occur in individuals when this mechanism is defective.
Figure 1. Localization of a SpII S * and FANCA in the nucleus of normal and FA-A cells after treatment with a DNA interstrand cross-linking agent, 8-MOP plus UVA light. (A) a SpII S * and FANCA co-localize in damaged normal cell nuclei, (B) decreased formation of a SpII S * foci and no FANCA foci in damaged FA-A cells, (C) a SpII S * and FANCA again co-localize in nuclear foci in 8-MOP plus UVA treated corrected FA-A cells.
Figure 2. Model for the role of a SpII S * in repair of DNA interstrand cross-links in (A) normal and (B) Fanconi anemia cells. In normal cells, FANC gene products regulate the stability of a SpII S *. a SpII S * binds to cross-linked DNA, acts as a scaffold and aids in the recruitment and alignment of repair proteins and FANC proteins to the site of damage thus enhancing the efficiency of the repair process. In FA cells, defects in the FANC genes or gene products lead to decreased levels of a SpII S * due to decreased stability of a SpII S *. Decreased levels of a SpII S * lead to decreased binding of this protein to damaged DNA and decreased recruitment and alignment of repair proteins at sites of damage, thus reducing the efficiency of DNA repair in FA cells.
Lambert, M.W., and Lambert, W.C. DNA repair and chromatin structure in genetic diseases. Prog. in Nucleic Acid Res. and Mol. Biol. 64:257-309, 1999.
Brois, D.W., McMahon, L.W., Ramos, N.I., Anglin, L.M., Walsh, C.E., and Lambert, M.W. A deficiency in a 230 kDa DNA repair protein in Fanconi anemia complementation group A cells is corrected by the FANCA cDNA. Carcingenesis, 20:1845-1853, 1999.
McMahon, L.W., Walsh, C.E., and Lambert, M.W. Human a spectrin II and the Fanconi anemia proteins FANCA and FANCC interact to form a nuclear complex. J. Biol. Chem., 274:32904-32908, 1999.
Kumaresan, K., and Lambert, M.W. Fanconi anemia, complementation group A, cells are defective in ability to produce incisions at sites of psoralen interstrand cross-links. Carcinogenesis, 21: 741-751, 2000.
Lambert, M.W., and Yang, L. Xeroderma pigmentosum complementation group A protein acts as a processivity factor. Biochem. Biophys. Res. Comm., 271:782-787, 2000.
McMahon, L.W., Sangerman, J., Goodman, S.R., Kumaresan, K., and Lambert, M.W. Human a spectrin II and the FANCA, FANCC, and FANCG proteins bind to DNA containing psoralen interstrand cross-links. Biochemistry, 40:7025-7034, 2001.
Kumaresan, K.R., Hwang, M., Thelen, M.P., and Lambert, M.W. Contribution of XPF functional domains to the 5' and 3' incisions produced at the site of a psoralen interstrand cross-link. Biochemistry, 41:890-896, 2002.
Sridharan, D., Brown, M., Lambert, W.C., McMahon, L.W., and Lambert, M.W. Nonerythroid a II spectrin is required for recruitment of FANCA and XPF to nuclear foci induced by DNA interstrand cross-links. J. Cell Science, 116:823-835, 2003.
Lefferts, J.A., and Lambert, M.W. Fanconi anemia cell lines deficient in a II spectrin express normal levels of a II spectrin mRNA. Biochem. Biophys. Res. Comm., 307:510-515, 2003.