Bookmark and Share

Patricia J. Gearhart Ph.D.

Academic Title: Adjunct Associate Professor
Primary Appointment: Microbiology and Immunology
Location: National Institute of Aging/National Institutes of Health
Phone: (410) 558-8561
Phone: (410) 558-8147
Fax: (410) 558-8157

Research Interests:

Somatic hypermutation of immunoglobulin genes occurs at a frequency that is a million times greater than mutation in other genes. Mutations are found in both variable genes and switch regions before constant genes. The molecular mechanism that introduces these mutations is intensely being studied. Hypermutation is initiated when the activation-induced cytidine deaminase (AID) protein deaminates cytosine in DNA to uracil, which causes C:G mutations. However, in B lymphocytes, substitutions of all four bases occur at similar levels, indicating that other proteins are required to generate mutations of A:T base pairs. We are studying how mismatch repair proteins and DNA polymerases are involved in the process. Mismatches are recognized by Msh2, Msh3, Msh6, Pms2, and Mlh1. We have examined hypermutation in mice deficient for all these proteins. All of the mice have hypermutation, but the types of substitutions vary. Mice deficient for Msh3, Pms2, and Mlh1 have relatively normal hypermutation, but mice deficient for Msh2 and Msh6 have a dramatic decrease in mutations of A and T, and an increase in mutations of C and G. Thus, the Msh2-Msh6 heterodimer may recognize the uracil lesion and recruit other proteins to cause mutations of downstream A and T nucleotides.

Mutations are actually put in by inaccurate DNA polymerases. We are studying the roles of DNA polymerases eta and iota in the mechanism. For polymerase eta, variable and switch regions were sequenced from patients with xeroderma pigmentosum variant disease, who are deficient in the polymerase. The frequency of hypermutation and heavy chain class switching was normal, but the types of base changes were different. Polymerase eta-deficient clones had a decrease in mutations at A and T with a concomitant rise of mutations at G and C. This finding implies that polymerase eta is an A-T mutator in hypermutation. Furthermore, there was an increase in C mutations on the nontranscribed strand in the switch regions, proving that stable secondary DNA structures preferentially expose unpaired cytosines for deamination by AID. For polymerase iota, variable genes were sequenced from the 129 strain of mice which are deficient in the polymerase. The frequency and pattern of hypermutation was similar to wildtype mice, indicating that its role is nonessential. To test for a dual knockout of polymerases eta and iota, we will examine mice that are deficient in both enzymes. Since Msh2-Msh6 and polymerase eta gave the same phenotype, that is, fewer A:T mutations, they may be interacting together at the uracil lesion. We are currently studying the biochemical and functional interactions of these and other proteins that have been implicated in this error-prone repair pathway. It may soon be possible to assemble all the pieces of the enigmatic hypermutation puzzle.


Martomo, S.A., Yang, W.W., and Gearhart, P.J. A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination. J. Exp. Med., in press.

Xeng, X., Negrete, G.A., Kasmer, C., Yang, W.W., and Gearhart, P.J. Absence of DNA polymerase eta reveals targeting of C mutations on the non-transcribed strand in immunoglobulin switch regions. J. Exp. Med. 199:917-924, 2004.

McDonald, J. P., Frank, E. G., Plosky, B. S., Rogozin, I. B., Masutani, C., Hanaoka, F., Woodgate, R., and Gearhart, P. J. 129-derived strains of mice are deficient in DNA polymerase iota ?and have normal immunoglobulin hypermutation. J. Exp. Med., in press.

Gearhart, P. J. B cells pay a price. Oncogene, in press.

Winter, D. B., Phung, Q. H., Zeng, X., Seeberg, E., Barnes, D. E., Lindahl, T., and Gearhart, P. J. Normal somatic hypermutation of immunoglobulin genes in the absence of 8-hydroxyguanine-DNA glycosylase. J. Immunol. 170:5558-5562, 2003.

Gearhart, P. J. The roots of antibody diversity. Nature 419:29-30, 2002.