TY - JOUR
T1 - Intrinsically disordered domain of tumor suppressor p53 facilitates target search by ultrafast transfer between different DNA strands
AU - Itoh, Yuji
AU - Murata, Agato
AU - Takahashi, Satoshi
AU - Kamagata, Kiyoto
N1 - Funding Information:
Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science [KAKENHI 16K07313 and 16KK0157 to K.K. and JP25104007 to S.T.]. Funding for open access charge: Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science [KAKENHI 16K07313]. Conflict of interest statement. None declared.
Funding Information:
Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science [KAKENHI 16K07313 and 16KK0157 to K.K. and JP25104007 to S.T.]. Funding for open access charge: Ministry of Education, Culture, Sports, Science and Technology/Japan Society for the Promotion of Science [KAKENHI 16K07313].
Publisher Copyright:
© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.
PY - 2018/8/21
Y1 - 2018/8/21
N2 - Intersegmental transfer (IST) is an important strategy in the target search used by sequence-specific DNA-binding proteins (DBPs), enabling DBPs to search for targets between multiple DNA strands without dissociation. We examined the IST of the tumor suppressor p53 using ensemble stopped-flow and single-molecule fluorescence measurements. The ensemble measurements demonstrated that p53 exhibits very fast IST, whose rate constant was ∼108 M−1 s−1. To determine the domains of p53 responsible for IST, two mutants with deletions of one of its two DNA binding domains were generated. The mutant lacking the disordered C-terminal (CT) domain (the CoreTet mutant) abolished IST, whereas the mutant lacking the structured core domain (the TetCT mutant) maintained IST, clearly demonstrating the importance of the CT domain. Single-molecule fluorescence measurements further demonstrated the transfer of p53 between two tethered DNA strands. The pseudo-wild-type p53 and the TetCT mutant showed significant transfer efficiencies, whereas the transfer efficiency for the CoreTet mutant was zero. These results suggest that ultrafast IST might be promoted by four copies of the CT domain, by binding to two DNA strands simultaneously. Such ultrafast IST might be important to avoid nearby-bound DBPs during the target search process of p53 in nucleus.
AB - Intersegmental transfer (IST) is an important strategy in the target search used by sequence-specific DNA-binding proteins (DBPs), enabling DBPs to search for targets between multiple DNA strands without dissociation. We examined the IST of the tumor suppressor p53 using ensemble stopped-flow and single-molecule fluorescence measurements. The ensemble measurements demonstrated that p53 exhibits very fast IST, whose rate constant was ∼108 M−1 s−1. To determine the domains of p53 responsible for IST, two mutants with deletions of one of its two DNA binding domains were generated. The mutant lacking the disordered C-terminal (CT) domain (the CoreTet mutant) abolished IST, whereas the mutant lacking the structured core domain (the TetCT mutant) maintained IST, clearly demonstrating the importance of the CT domain. Single-molecule fluorescence measurements further demonstrated the transfer of p53 between two tethered DNA strands. The pseudo-wild-type p53 and the TetCT mutant showed significant transfer efficiencies, whereas the transfer efficiency for the CoreTet mutant was zero. These results suggest that ultrafast IST might be promoted by four copies of the CT domain, by binding to two DNA strands simultaneously. Such ultrafast IST might be important to avoid nearby-bound DBPs during the target search process of p53 in nucleus.
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U2 - 10.1093/nar/gky586
DO - 10.1093/nar/gky586
M3 - Article
C2 - 29986056
AN - SCOPUS:85052843832
VL - 46
SP - 7261
EP - 7269
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 14
ER -