 |
Patricia J. Gearhart, Ph.D., Senior Investigator Section on Antibody Diversity Laboratory of Molecular Gerontology Phone: 410-558-8561 Fax: 410-558-8157 E-mail: gearhartp@grc.nia.nih.gov |
| Biography: Dr. Patricia Gearhart received her Ph.D. in immunology from the University of Pennsylvania in 1974. She performed postdoctoral training at the Johns Hopkins University and was a staff associate at the Carnegie Institution of Washington until 1982. She then became a faculty member at the Johns Hopkins University until 1995, when she moved to the NIA. |
| Proteins Involved in Somatic Hypermutation of Immunoglobulin Variable Genes: 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. We have used biochemical and genetic techniques to study this fascinating process that occurs constantly in every B lymphocyte in the human body. Hypermutation is initiated when the activation-induced cytidine deaminase (AID) protein deaminates cytosine in DNA to uracil. First, we demonstrated that uracil is present in the DNA of bacterial cells that express the AID protein. Using quantitative Southern and ligation-mediated PCR techniques, uracils were detected in actively transcribed genes. The uracils were made predominantly on the nontranscribed strand, in accord with the notion that AID acts on single-stranded DNA. We now plan to detect uracils in variable and switch regions in B cells undergoing mutation. Second, we studied the spectra of mutations in mice lacking mismatch repair proteins and low-fidelity DNA polymerases. Mice deficient for the Msh2-Msh6 heterodimer and DNA polymerase eta have fewer mutations of A:T base pairs, whereas mutations of C:G base pairs can be directly caused by replication or repair of the uracils. Third, using biochemical techniques we demonstrated that the uracil base is recognized by Msh2-Msh6, which then recruits DNA polymerase eta and stimulates its catalytic activity. Thus, synthesis of DNA by this error-prone polymerase can generate mutations downstream of the uracils, including mutations at A:T base pairs. We also showed that polymerase eta is responsible for synthesizing mutations in a repair patch on the non-transcribed strand, and generates more mutations of A than T, in accord with the spectra observed in vivo. |
| Targeting of AID to Immunoglobulin Genes: A major question in antibody diversity concerns how hypemutation is targeted to the immunoglobulin loci, and is not found elsewhere in DNA from B cells. Mutations are localized to two distinct regions: two kilobases of DNA surrounding and including the rearranged variable gene, and four kilobases of DNA encompassing the switch regions. These regions are downstream of promoters, suggesting that transcription is involved in bringing AID to the locus. To address this question, we will study how transcription proteins interact with the various enzymes involved in the hypermutation pathway. It soon may be possible to assemble all the pieces of the enigmatic hypermutation puzzle. |
|
 |
| Section on Antibody Diversity (left to right). Row 1: Patricia Gearhart, Deepa Rajagopal, Robert Maul; row 2: William Yang, Nicholas Durham, Huseyin Saribasak. |
| IRP Home What's New Contact Us Accessibility Disclaimer Privacy Site Search Site Map NIA Home |
 |
 |
 |