Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination.
The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.
Repasky
Jamie A E
JA
Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06510, USA.
Corbett
Elizabeth
E
Boboila
Cristian
C
Schatz
David G
DG
D000818:Animals; D000916:Antibody Diversity / Q000235:genetics*; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004252:DNA Mutational Analysis; D004253:DNA Nucleotidylexotransferase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015321:Gene Rearrangement / Q000276:immunology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007133:Immunoglobulin Joining Region / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins; D009711:Nucleotides / Q000235:genetics / Q000378:metabolism*; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic / Q000276:immunology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013379:Substrate Specificity / Q000235:genetics; D013698:Templates, Genetic
D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007527:Isoenzymes; D009687:Nuclear Proteins; D009711:Nucleotides; C499311:RAG2 protein, human; C499308:Rag2 protein, mouse; C064655:V(D)J recombination activating protein 2; C064658:RAG-1 protein; D004253:DNA Nucleotidylexotransferase