Abstract

This proposal is to examine breaks in DNA that occur during the process of immunoglobulin class switch recombination (CSR). CSR is the process by which B lymphocytes exchange the constant region of the immunoglobulin (Ig) molecule they produce in order to most effectively combat the pathogen to which they have been exposed. CSR involves a recombination event in which the IgM constant region gene is deleted and replaced by a downstream constant region gene such as C?. The recombination occurs within DNA located upstream of each constant region gene known as switch (S) regions. As the intervening DNA is excised as a circle, the process requires that double strand breaks are made in both the upstream and downstream S regions. DNA breaks can lead to mutations, translocations and tumors and therefore must be tightly regulated. It is known that activation-induced cytidine deaminase (AID) is required for this process and that it can deaminate cytidines in DNA to generate uracils that can be mutagenic and/or lead to DNA breaks. The experiments proposed here will determine if AID acts directly on Ig S region DNA in vivo to convert cytidines to uracils and also will determine the subsequent steps that lead to break formation. Ligation-mediated (LM)-PCR will be used to detect the exact breakpoint in S region DNA and to test the hypothesis that the breaks instigated by AID activity are initially single-stranded and staggered double strand breaks (DSBs), but that end-processing by DNA repair enzymes can convert some of these breaks to blunt DSBs. The position and structure of breaks will be examined in cells from mice deficient in DNA repair proteins from the mismatch repair (MMR), nucleotide excision repair (NER), and base excision repair (BER) pathways. The BER enzymes UNG and APE can remove uracil from DNA and nick the DNA backbone and are thought to be involved in break formation. Enzymes from the BER pathway will be used to treat genomic DNA from B cells induced to switch in order to detect intermediates in the repair pathway that are predicted by this model.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI065639-04
Application #
7373526
Study Section
Cellular and Molecular Immunology - B (CMI)
Program Officer
Nasseri, M Faraz
Project Start
2005-05-15
Project End
2010-01-31
Budget Start
2008-02-01
Budget End
2009-01-31
Support Year
4
Fiscal Year
2008
Total Cost
$264,514
Indirect Cost

  Institution

Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655

  Publications

Stavnezer, Janet; Linehan, Erin K; Thompson, Mikayla R et al. (2014) Differential expression of APE1 and APE2 in germinal centers promotes error-prone repair and A:T mutations during somatic hypermutation. Proc Natl Acad Sci U S A 111:9217-22
Guikema, Jeroen E J; Linehan, Erin K; Esa, Nada et al. (2014) Apurinic/apyrimidinic endonuclease 2 regulates the expansion of germinal centers by protecting against activation-induced cytidine deaminase-independent DNA damage in B cells. J Immunol 193:931-9
Guikema, Jeroen E J; Gerstein, Rachel M; Linehan, Erin K et al. (2011) Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge. J Immunol 186:1943-50
Pei, De-Sheng; Yang, Xiao-Jie; Liu, Wei et al. (2011) A novel regulatory circuit in base excision repair involving AP endonuclease 1, Creb1 and DNA polymerase beta. Nucleic Acids Res 39:3156-65
Guikema, Jeroen E J; Schrader, Carol E; Brodsky, Michael H et al. (2010) p53 represses class switch recombination to IgG2a through its antioxidant function. J Immunol 184:6177-87
Eccleston, Jennifer; Schrader, Carol E; Yuan, Karen et al. (2009) Class switch recombination efficiency and junction microhomology patterns in Msh2-, Mlh1-, and Exo1-deficient mice depend on the presence of mu switch region tandem repeats. J Immunol 183:1222-8
Guikema, Jeroen E J; Schrader, Carol E; Leus, Niek G J et al. (2008) Reassessment of the role of Mut S homolog 5 in Ig class switch recombination shows lack of involvement in cis- and trans-switching. J Immunol 181:8450-9
Stavnezer, Janet; Guikema, Jeroen E J; Schrader, Carol E (2008) Mechanism and regulation of class switch recombination. Annu Rev Immunol 26:261-92
Schrader, Carol E; Guikema, Jeroen E J; Linehan, Erin K et al. (2007) Activation-induced cytidine deaminase-dependent DNA breaks in class switch recombination occur during G1 phase of the cell cycle and depend upon mismatch repair. J Immunol 179:6064-71
Guikema, Jeroen E J; Linehan, Erin K; Tsuchimoto, Daisuke et al. (2007) APE1- and APE2-dependent DNA breaks in immunoglobulin class switch recombination. J Exp Med 204:3017-26

Showing the most recent 10 out of 12 publications