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 modifying a genetic substance of a cell, the method comprising:
 designing a DNA binding protein;   determining a DNA base sequence coding for an amino acid sequence of a fused protein comprising a functional region and a DNA binding region consisting of a DNA-binding protein;   cloning said DNA base sequence;   preparing a vector carrying said DNA base sequence and a cell containing a DNA having at least 9 target DNA bases as a target DNA base sequence; and   introducing the vector into the cell so that the DNA binding region of the fused protein binds to the DNA having the at least 9 target DNA bases, and therefore the functional region modifies the DNA having the at least 9 target DNA bases,   wherein the DNA-binding protein contains at least 9 PPR motifs for binding to the at least 9 target DNA bases, respectively, and 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, 
 position numbers of amino acids in the PPR motifs are the same as PF01535 in Pfam 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), 
 
 each PPR motif (M n ) contained in the DNA-binding protein is a PPR motif having a specific combination of amino acids as the three amino acids of Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, such that the DNA-binding protein has at least 9 combinations of the three amino acids corresponding to the respective target DNA bases of the target DNA base sequence, and each combination of the three amino acids is determined according to any one of the following definitions: 
 (2-1) when the target DNA base to which the PPR motif binds is 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; 
 (2-2) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are glutamic acid, glycine, and aspartic acid, respectively; 
 (2-3) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, glycine, and asparagine, respectively; 
 (2-4) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are glutamic acid, glycine, and asparagine, respectively; 
 (2-5) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, glycine, and serine, respectively; 
 (2-7) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, isoleucine, and asparagine, respectively; 
 (2-12) when the target DNA base to which the PPR motif binds is T, 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; 
 (2-13) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, methionine, and aspartic acid, respectively; 
 (2-15) when the target DNA base to which the PPR motif binds is T, 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; 
 (2-16) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, asparagine, and aspartic acid, respectively; 
 (2-17) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are glycine, asparagine, and aspartic acid, respectively; 
 (2-18) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, asparagine, and aspartic acid, respectively; 
 (2-19) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are threonine, asparagine, and aspartic acid, respectively; 
 (2-20) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA are valine, asparagine, and aspartic acid, respectively; 
 (2-21) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA are tyrosine, asparagine, and aspartic acid, respectively; 
 (2-22) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and asparagine, respectively; 
 (2-23) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, asparagine, and asparagine, respectively; 
 (2-24) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are serine, asparagine, and asparagine, respectively; 
 (2-25) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, asparagine, and asparagine, respectively; 
 (2-26) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and serine, respectively; 
 (2-27) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, asparagine, and serine, respectively; 
 (2-28) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and threonine, respectively; 
 (2-29) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, asparagine, and threonine, respectively; 
 (2-30) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, asparagine, and tryptophan, respectively; 
 (2-31) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, asparagine, and tryptophan, respectively; 
 (2-33) when the target DNA base to which the PPR motif binds is T, 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; 
 (2-34) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, proline, and aspartic acid, respectively; 
 (2-35) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are tyrosine, proline, and aspartic acid, respectively; 
 (2-37) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, serine, and asparagine, respectively; 
 (2-38) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, serine, and asparagine, respectively; 
 (2-39) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, serine, and asparagine, respectively; 
 (2-41) when the target DNA base to which the PPR motif binds is 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; 
 (2-42) when the target DNA base to which the PPR motif binds is the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, threonine, and aspartic acid, respectively; 
 (2-43) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, threonine, and asparagine, respectively; 
 (2-44) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, threonine, and asparagine, respectively; 
 (2-45) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, threonine, and asparagine, respectively; 
 (2-46) when the target DNA base to which the PPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine, threonine, and asparagine, respectively; 
 (2-48) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are isoleucine, valine, and aspartic acid, respectively; 
 (2-49) when the target DNA base to which the PPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, valine, and glycine, respectively; and 
 (2-50) when the target DNA base to which the PPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid, valine, and threonine, respectively. 
 
     
     
         2 . The method according to  claim 1 , wherein the functional region is fused to the DNA-binding protein on the C-terminus side of the protein. 
     
     
         3 . The method according to  claim 1 , 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. 
     
     
         4 . The method according to  claim 3 , wherein the DNA-cleaving enzyme is the nuclease domain of FokI (SEQ ID NO: 6). 
     
     
         5 . The method according to  claim 1 , wherein the one or more PPR motifs are any group of motifs 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.