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Antibody Diversity Section Patricia J. Gearhart, Ph.D., Senior Investigator |
| 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. |
| AID and Somatic Hypermutation: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 deaminase (AID) protein deaminates cytosine in DNA to uracil. |
| Switch Sequence Causes RNA Polymerase II Accumulation: We examined how DNA structure affects transcription and hypermutation, to target AID to the switch region. The position of RNA polymerase II molecules and mutations was mapped across a 5 kb region spanning the intronic enhancer to the constant m gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)-deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1 kb repetitive sequence that forms the core of the switch region. For mutations, DNA was sequenced from Ung-/- B cells stimulated in vivo, and mutations increase dramatically in the switch region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and allows AID access to single strand DNA. We are currently determining if polymerases are paused in the variable region, and what DNA sequences cause pausing. |
| AID-Dependent Uracils in Immunoglobulin Variable and Switch Regions: Two opposing models have been proposed for AID to deaminate either DNA or RNA. Although most data supports DNA deamination, there is no physical evidence for uracils in immunoglobulin genes. We have demonstrated their presence by determining the sensitivity of DNA to digestion with UNG and abasic endonuclease. Using several methods of detection, we identified uracils in the variable and switch regions. Uracils were generated within 24 hours after B cell stimulation, were present on both DNA strands, and were found predominantly at cytosine bases.This data provides direct evidence for the model that AID functions by deaminating cytosine in DNA. |
| Competition between DNA Repair and Mutagenesis: Uracils in DNA can be recognized by UNG and abasic endonuclease to produce single-strand breaks. The breaks are either repaired faithfully by DNA base excision repair (BER), or mutagenically to produce somatic hypermutation and class switch recombination. To unravel the interplay between repair and mutagenesis, we decreased the level of X-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of mutation in Igh variable regions and double-strand breaks in the switch regions during recombination. Although the frequency of switching was normal in Xrcc1+/- B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative-nonhomologous end joining. Furthermore, Xrcc1+/- B cells had reduced Igh/c-myc translocations, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways. |
PubMed: Search for listing of Dr. Gearhart's publications. |
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Antibody Diversity Section |
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