Terminal deoxynucleotidyltransferase forms a ternary complex with a novel chromatin remodeling protein with 82 kDa and core histone.
BACKGROUND
Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances the Ig and TcR gene diversity in the N region at the junctions of variable (V), diversity (D) and joining (J) segments in B- and T-cells. TdT synthesizes the N region in concert with many proteins including DNA-PKcs, Ku70 and Ku86. To elucidate the molecular mechanism of the N region synthesis, we first attempted to isolate the genes with products that directly interact 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 2 (TdIF2). The confined region of the C-terminal in TdIF2 is involved in specific interaction with the entire C-terminal in TdT. TdIF2 contains an acidic region comprised of 42 residues. TdIF2 was shown to bind specifically to a core histone by pull down assay using specific antibodies against TdIF2. When a TdT/TdIF2 complex was applied on to a DNA-cellulose column, only TdT bound to the column while TdIF2 passed through. TdIF2 reduces the TdT activity to 46% of its maximum value in vitro assay system using activated DNA as primer.


CONCLUSIONS
TdIF2 binds directly to TdT and core histone. Furthermore, TdT, TdIF2 and core histone form a ternary complex. TdIF2 liberates H2A/H2B from a core histone in correlation with PCNA. The enzymatic consequence of the TdIF2/TdT complex is the reduction of TdT activity in vitro. TdIF2 would function as a chromatin remodeling protein at the N region synthesis.
Fujita
Kiyoko
K
Faculty of Science and Technology, Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
Shimazaki
Noriko
N
Ohta
Yuko
Y
Kubota
Takashi
T
Ibe
Shirou
S
Toji
Shingo
S
Tamai
Katsuyuki
K
Fujisaki
Seiichiro
S
Hayano
Takahide
T
Koiwai
Osamu
O
D000595:Amino Acid Sequence; D002482:Cellulose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques
D002843:Chromatin; C033190:DNA-cellulose; D006657:Histones; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D002482:Cellulose; D004247:DNA; D004253:DNA Nucleotidylexotransferase