Identification of functional domains in TdIF1 and its inhibitory mechanism for TdT activity.
TdT interacting factor 1 (TdIF1) was identified as a protein that binds to terminal deoxynucleotidyltransferase (TdT) to negatively regulate TdT activity. TdT is a template-independent DNA polymerase that catalyzes the incorporation of deoxynucleotides to the 3'-hydroxyl end of DNA templates to increase the junctional diversity of immunoglobulin or T-cell receptor (TcR) genes. Here, using bioinformatics analysis, we identified the TdT binding, DNA binding and dimerization regions, and nuclear localization signal (NLS) in TdIF1. TdIF1 bound to double-stranded DNA (dsDNA) through three DNA binding regions: residues 1-75, the AT-hook-like motif (ALM) and the predicted helix-turn-helix (HTH) motif. ALM in TdIF1 preferentially bound to AT-rich DNA regions. NLS was of the bipartite type and overlapped ALM. TdIF1 bound to the Pol beta-like region in TdT and blocked TdT access to DNA ends. In the presence of dsDNA, however, TdIF1 bound to dsDNA to release TdT from the TdIF1/TdT complex and to exhibit TdT activity, implying that active TdT released microenvironmentally concentrates around AT-rich DNA to synthesize DNA.
Kubota
Takashi
T
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba 278-8510, Japan.
Maezawa
So
S
Koiwai
Kotaro
K
Hayano
Takahide
T
Koiwai
Osamu
O
D020856:AT Rich Sequence; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D004253:DNA Nucleotidylexotransferase / Q000037:antagonists & inhibitors*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D019281:Dimerization; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C436745:DNTTIP1 protein, human; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D014157:Transcription Factors; C109534:DNA polymerase X; D004253:DNA Nucleotidylexotransferase; D004259:DNA-Directed DNA Polymerase