Dna-binding protein using ppr motif, and use thereof

Abstract

The object of the present invention is to, by analyzing PPR proteins that act to bind to DNA with a prediction that RNA recognition rules of PPR motifs can also be used for recognition of DNA, find a PPR protein showing such a characteristic. According to the present invention, it was revealed that, with a protein that can bind in a DNA base-selective manner or a DNA base sequence-specific manner, which contains one or more, preferably 2 to 30, more preferably 5 to 25, most preferably 9 to 15, of PPR motifs having a structure of the following formula 1 (wherein, in the formula 1, Helix A is a part that can form an α-helix structure; X does not exist, or is a part consisting of 1 to 9 amino acids; Helix B is a part that can form an α-helix structure; and L is a part consisting of 2 to 7 amino acids), and having a specific combination of amino acids corresponding to a DNA base or DNA base sequence as amino acids of three positions of No. 1 A.A., No. 4 A.A., in Helix A of the formula 1 and No. “ii” (−2) A.A. contained in L of the formula 1, the aforementioned object could be achieved. (Helix A )-X-(Helix B )-L  (Formula 1)

What is claimed is: 
     
         1 . A method for preparing a protein that can bind in a DNA base-selective manner or a DNA base sequence-specific manner, comprising:
 providing a nucleic acid sequence encoding a protein;   cloning said nucleic acid sequence; and   preparing a transformant which produces the protein,   wherein the protein contains one or more PPR motifs having a structure of the following formula 1:
   (Helix  A )-X-(Helix  B )-L  (Formula 1)
 
   (wherein, in the formula 1:   Helix A is a part that can form an α-helix structure;   X does not exist, or is a part consisting of 1 to 9 amino acids;   Helix B is a part that can form an α-helix structure; and   L is a part consisting of 2 to 7 amino acids),   wherein,   under the following definitions:   the first amino acid of Helix A is referred to as Number 1 amino acid (Number 1 AA), the fourth amino acid as Number 4 amino acid (Number 4 AA), and
 when a next PPR motif (M n+1 ) contiguously exists on the C-terminus side of the PPR motif (M n ) (when there is no amino acid insertion between the PPR motifs), the −2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M n ); 
 when a non-PPR motif consisting of 1 to 20 amino acids exists between the PPR motif (M n ) and the next PPR motif (M n+1 ) on the C-terminus side, the amino acid locating upstream of the first amino acid of the next PPR motif (M n+1 ) by 2 positions, i.e., the −2nd amino acid; or 
 when any next PPR motif (M n+1 ) does not exist on the C-terminus side of the PPR motif (M n ), or 21 or more amino acids constituting a non-PPR motif exist between the PPR motif (M n ) and the next PPR motif (M n+1 ) on the C-terminus side, the 2nd amino acid counted from the end (C-terminus side) of the amino acids constituting the PPR motif (M n ) 
   is referred to as Number “ii” (−2) amino acid (Number “ii” (−2) AA),   one PPR motif (M n ) contained in the protein is a PPR motif having a specific combination of amino acids corresponding to a target DNA base or target DNA base sequence as the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (−2) AA.   
     
     
         2 . The method according to  claim 1 , wherein the combination of the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (−2) AA is a combination corresponding to a target DNA base or target DNA base sequence, and the combination of amino acids is determined according to any one of the following definitions:
 (1-1) when Number 4 AA is glycine (G), Number 1 AA may be an arbitrary amino acid, and Number “ii” (−2) AA is aspartic acid (D), asparagine (N), or serine (S); 
 (1-2) when Number 4 AA is isoleucine (I), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; 
 (1-3) when Number 4 AA is leucine (L), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; 
 (1-4) when Number 4 AA is methionine (M), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; 
 (1-5) when Number 4 AA is asparagine (N), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; 
 (1-6) when Number 4 AA is proline (P), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; 
 (1-7) when Number 4 AA is serine (S), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; 
 (1-8) when Number 4 AA is threonine (T), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid; and 
 (1-9) when Number 4 AA is valine (V), each of Number 1 AA and Number “ii” (−2) AA may be an arbitrary amino acid. 
 
     
     
         3 . The method according to  claim 1 , wherein the combination of the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (−2) AA is a combination corresponding to a target DNA base or target DNA base sequence, and the combination of amino acids is determined according to any one of the following definitions:
 (2-1) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA are an arbitrary amino acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G; 
 (2-2) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are glutamic acid, glycine, and aspartic acid, respectively, the PPR motif selectively binds to G; 
 (2-3) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-4) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are glutamic acid, glycine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-5) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, glycine, and serine, respectively, the PPR motif selectively binds to A, and next binds to C; 
 (2-6) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, isoleucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C; 
 (2-7) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, isoleucine, and asparagine, respectively, the PPR motif selectively binds to T, and next binds to C; 
 (2-8) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, leucine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T and C; 
 (2-9) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, leucine, and aspartic acid, respectively, the PPR motif selectively binds to C; 
 (2-10) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, leucine, and lysine, respectively, the PPR motif selectively binds to T; 
 (2-11) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, methionine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T; 
 (2-12) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-13) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, methionine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; 
 (2-14) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to C and T; 
 (2-15) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-16) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-17) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are glycine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-18) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-19) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are threonine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-20) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA are valine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; 
 (2-21) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA are tyrosine, asparagine, and aspartic acid, respectively, the PPR motif selectively binds to T, and next binds to C; 
 (2-22) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; 
 (2-23) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; 
 (2-24) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are serine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; 
 (2-25) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, asparagine, and asparagine, respectively, the PPR motif selectively binds to C; 
 (2-26) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and serine, respectively, the PPR motif selectively binds to C; 
 (2-27) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, asparagine, and serine, respectively, the PPR motif selectively binds to C; 
 (2-28) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and threonine, respectively, the PPR motif selectively binds to C; 
 (2-29) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, asparagine, and threonine, respectively, the PPR motif selectively binds to C; 
 (2-30) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, asparagine, and tryptophan, respectively, the PPR motif selectively binds to C, and next binds to T; 
 (2-31) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, asparagine, and tryptophan, respectively, the PPR motif selectively binds to T, and next binds to C; 
 (2-32) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, proline, and an arbitrary amino acid, respectively, the PPR motif selectively binds to T; 
 (2-33) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-34) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-35) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are tyrosine, proline, and aspartic acid, respectively, the PPR motif selectively binds to T; 
 (2-36) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, serine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G; 
 (2-37) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, serine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-38) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, serine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-39) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, serine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-40) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, threonine, and an arbitrary amino acid, respectively, the PPR motif selectively binds to A and G; 
 (2-41) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G; 
 (2-42) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, threonine, and aspartic acid, respectively, the PPR motif selectively binds to G; 
 (2-43) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, threonine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-44) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are phenylalanine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-45) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-46) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are valine, threonine, and asparagine, respectively, the PPR motif selectively binds to A; 
 (2-47) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, valine, and an arbitrary amino acid, respectively, the PPR motif binds with A, C, and T, but does not bind to G; 
 (2-48) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are isoleucine, valine, and aspartic acid, respectively, the PPR motif selectively binds to C, and next binds to A; 
 (2-49) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, valine, and glycine, respectively, the PPR motif selectively binds to C; and 
 (2-50) when the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (−2) AA, are an arbitrary amino acid, valine, and threonine, respectively, the PPR motif selectively binds to T. 
 
     
     
         4 . The method according to  claim 1 , wherein the one or more PPR motifs are any selected from
 9 PPR motifs belonging to the p63 protein consisting of the amino acid sequence of SEQ ID NO: 1,   11 PPR motifs belonging to the GUN1 protein consisting of the amino acid sequence of SEQ ID NO: 2,   15 PPR motifs belonging to the pTac2 protein consisting of the amino acid sequence of SEQ ID NO: 3,   10 PPR motifs belonging to the DG1 protein consisting of the amino acid sequence of SEQ ID NO: 4, and   11 PPR motifs belonging to the GRP23 protein consisting of the amino acid sequence of SEQ ID NO: 5.   
     
     
         5 . A method for controlling a function of DNA, the method comprising:
 preparing a DNA-binding protein by the method according to  claim 1 , and   allowing the protein to bind to a DNA molecule having a target DNA base or target DNA base sequence to control a function of the DNA molecule.   
     
     
         6 . A method for modifying a genetic substance of a cell, the method comprising:
 preparing a DNA-binding protein by the method according to  claim 1  as a fused protein comprising a functional region and a DNA binding region consisting of the DNA-binding protein;   preparing a cell containing a DNA having a target sequence; and   introducing the fused protein into the cell so that the DNA binding region of the fused protein binds to the DNA having a target sequence, and therefore the functional region modifies the DNA having a target sequence.   
     
     
         7 . The method according to  claim 6 , wherein the functional region is fused to the DNA-binding protein on the C-terminus side of the protein. 
     
     
         8 . The method according to  claim 6 , wherein the functional region is a DNA-cleaving enzyme, or a nuclease domain thereof, or a transcription control domain, and the complex functions as a target sequence-specific DNA-cleaving enzyme or transcription control factor. 
     
     
         9 . The method according to  claim 8 , wherein the DNA-cleaving enzyme is the nuclease domain of FokI (SEQ ID NO: 6).