The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes takes place in B lymphocytes after specific interaction with antigen and T lymphocytes. SHM has several steps and requirements. In our working model, the cell needs to produce a postulated mutator factor (MuF) which has to be targeted to Ig genes by transcription. The MuF may act as an endonuclease that creates a nick or double-strand break in the gene. The nick/break is believed to be repaired in an error-prone fashion, creating mutations. Mismatch repair may remove some of the mutations or fix them by "correcting" the wildtype nucleotide on the complementary DNA strand. Cell replication propagates the mutations to one or both daughter cells. The following investigations are proposed: 1) What is the role of a specific E-box site in SHM? Such a transctivator site was found to enhance SHM without enhancing transcription. 2) How does the position of nucleosomes affect the SHM process? SHM occurs in clusters that suggest involvement of nucleosomes in the targeting of mutations. Mono-nucleosomal DNA from a mutable mouse transgene will be tested for phasing of nucleosomes over the SHM target relative to mutations. 3) Can a double-strand DNA break induce SHM? The possibility will be tested that introduction of a double strand break into a SHM target gene induces mutations without the need for the initiating events of SHM. 4) When during the cell cycle does SHM occur? It is still unknown if DNA replication of the genome is involved directly in SHM, and/or if recombination is required. Single cells will be isolated at various cell cycle stages and screened for molecular intermediates consistent with an ongoing mutation process. 5) Which mRNAs are modified by the cytidine deaminase, AID, in mutating B lymphocytes? The cytosine deaminase, AID, is required for SHM. The potential target mRNAs will be identified. The planned experiments are important for learning how the varied repertoire of Ig genes is created with the potential to react against any foreign antigenic determinant, including tumor cell antigens. Somatic hypermutation has also been implicated in autoimmune diseases. Furthermore, many B cell lymphomas arise apparently as a consequence of the somatic mutation process. It is likely that understanding the components involved in somatic mutation will aid in understanding the genetic and environmental causes of autoimmunity, and the treatment of infectious diseases and tumors. Surprisingly, the BCL6 proto-oncogene is highly mutated in human memory B cells. This is likely to be involved in tumorigenesis. Thus, a better understanding of the somatic mutation process may aid in the understanding and perhaps prevention, diagnosis and treatment of human B lymphomas related to BCL6 expression. PERFORMANCESITE(S) (organizationc,ity,state) The University of Chicago, Chicago, Illinois KEYPERSONNELS. eeinstructionsU. secontinuationpagesasneededtoprovidetherequiredinformatiointheformatshownbelow. StartwithPrincipalInvestigatorL. istallotherkeypersonneiln alphabeticaolrder,lastnamefirst. Name Organization RoleonProject Ursula B. Storb The University of Chicago Principal Investigator Randal Cox None Consultant Peter Engler The University of Chicago Research Associate Stephen Gasior The University of Chicago Research Associate Simone Longerich The University of Chicago Grad Res Assistant Nancy Michael The University of Chicago Research Associate Hong Ming Shen The University of Chicago Research Associate DisclosurePermission StatemenL ApplicabletoSBIR/STTROnly.Seeinstructions[.] Yes [] No [] PHS398(Rev.05/01) Page 2 FormPage2 [] _h,mhepr agesconsec,,tivela,,tthebottomtr_ro,,gho,t,hte applicationDonot,,ses,,ffi,,es,,chas"aa.,ah [] PrincipalInvestigator/ProgramDirector(Last,first, middle): Storb, Ursula The name of theprincipalinvestigator/programdirectormustbe providedatthetop of eachprintedpage andeachcontinuationpage. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ............................................................................................................................................ 1 Description,

R01AI053130

2002-12-01

2007-11-30