Analysis of the role of the recombination signal sequence in the fidelity of V(D)J recombination.
Published: 2004
Language: eng
Number of pages: 222
Description: B and T lymphocytes assemble antigen receptor genes through a series of DNA rearrangements that target DNA recombination signal sequences (RSSs). This process, termed V(D)J recombination, plays a central role in lymphocyte development and the generation of a diverse immune repertoire of B cell immunoglobulins (Ig) and T cell receptors (TCR). While it is a critical operation, the recombination also poses tremendous risks. Aberrant rearrangement can contribute to the development of various lymphoid malignancies. To gain insight into the basis of V(D)J recombination specificity, I have investigated whether incorrectly targeted DNA sequences can be detected after they have been cleaved by RAG proteins. Using a murine extrachromosomal recombination system, I have observed that sequences incorrectly targeted and cleaved are not as efficiently rejoined as are authentic RSSs, indicating that sequence specificity exists beyond cleavage. Furthermore, these sequence requirements differ from those for binding and cleavage. In another study, I wished to learn how the RSS permits unusual rearrangement at the chicken immunoglobulin locus. I revealed a potential role for the RSS spacer sequence, which is the component of the RSS that was previously thought not to contribute to the specificity of V(D)J recombination. Rearrangement is mediated by the pairing of RSSs, one of which has a 12-bp spacer (12-RSS) and one of which has a 23-bp spacer (23-RSS), a feature known as the 12/23 rule. I introduced the chicken sequences into the murine recombination system and observed that the 12/23 rule can be violated to permit 12/12 rearrangement outside of the chicken. After performing sequence alignments of human and murine spacers and determining consensus sequences, I propose that the spacer sequence is a factor in RSS targeting. My research has extensively examined the role of the RSS in supporting efficient V(D)J recombination. I have provided evidence that unusual rearrangement at the chicken IgH locus is mediated by RSSs, and not by chicken-specific proteins. Furthermore, I have contributed  in vivo support for a V(D)J recombination post-cleavage complex involving the RAG proteins, suggesting that a late-stage consequence of DNA mistargeting is the interruption of recombination and/or reduced cellular survival.
Notes: Adviser: Susanna Lewis.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-05, Section: B, page: 2327.