Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65.
BACKGROUND
Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B- and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT.


RESULTS
Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG-I and HMG-Y DNA binding domains (AT-hooks) and can bind to single- and double-stranded DNA. TdT and TdIF1 were co-eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers.


CONCLUSIONS
TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.
Yamashita
N
N
Faculty of Science & Technology, Department of Applied Biological Science, Science University of Tokyo, Noda, Chiba 278-8510, Japan.
Shimazaki
N
N
Ibe
S
S
Kaneko
R
R
Tanabe
A
A
Toyomoto
T
T
Fujita
K
K
Hasegawa
T
T
Toji
S
S
Tamai
K
K
Yamamoto
H
H
Koiwai
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification*; D002874:Chromosome Mapping; D004253:DNA Nucleotidylexotransferase / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016328:NF-kappa B; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000737:chemistry*; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C436745:DNTTIP1 protein, human; C499052:Dnttip1 protein, mouse; D016328:NF-kappa B; D009687:Nuclear Proteins; D020033:Protein Isoforms; D018160:Receptors, Cytoplasmic and Nuclear; D051996:Transcription Factor RelA; D014157:Transcription Factors; D004253:DNA Nucleotidylexotransferase