A novel four zinc-finger protein targeted against p190BcrAbl fusion oncogene cDNA: utilisation of zinc-finger recognition codes

Abstract

A three zinc-finger protein that binds specifically to the cDNA representing the unique fusion gene BCR:Abl, associated with acute lymphoblastic leukaemia, has previously been characterised. At this breakpoint, a sequence homology of 8/9 bp exists between the BCR:Abl (fusion) and c-ABL: (parental) target sequences. We show that the three zinc-finger protein discriminates poorly between the fusion (BCR:Abl) and parental (ABL:) sequence (K:(d)s of 42.8 and 65.1 nM, respectively). In order to improve the discriminatory properties of this protein, and to demonstrate the utility of current zinc-finger databases, we have added a fourth zinc-finger to the original three zinc-finger protein. This fourth finger recognises a 3 bp subsite derived from the BCR: portion of the breakpoint and is not present in c-ABL: This novel four finger protein, which now recognises a 12 bp sequence, demonstrates improved specific binding to BcrAbl (K:(d )= 17 nM). More significantly we have shown that there is now enhanced discrimination between BcrAbl and ABL: sequences by the four finger protein than the original three finger protein.

Modulation of P2X3 and P2X2/3 Receptors by Monoclonal Antibodies

“…Bcr-Abl three-zinc finger protein (17) modified by a single zinc finger extension (12,13). Zinc finger peptides were expressed as N-terminal fusions with the HpaII (FH) mutant Mtase and were linked by a flexible linker (Gly 4 Ser) 3 .…”

“…One potential problem with these enzymes has been the level of associated nontargeted methylation, which is sometimes unacceptably high due to the avidity of the wild-type Mtase components used and is often overlooked. Recently we showed that, employing four zinc finger arrays capable of binding to chromatin target sites (12,13) in combination with reduced affinity/activity prokaryotic DNA cytosine methyltransferase mutants, methylation could be targeted to predetermined sequences with virtually no nonspecific methylation occurring (14). The HpaII methyltransferase F38H mutant used in these latter studies (henceforth referred to as FH) dem-onstrated a significantly overall reduced methyltransferase activity relative to the wild-type enzyme because of a mutation in the conserved FXGXG motif involved in Mtase cofactor binding and target base interaction, which allowed the zinc finger protein component to dominate in DNA-protein interactions.…”

Modified Transposases for Site-Directed Insertion of Transgenes

“…Attempts to target transposition in E. coli were made by fusing the N-terminus of Mos1 transposase to a four zinc fi nger DBD (ZF) (Demattéi et al 2010 ) that recognizes the bcr-abl fusion oncogene sequence (ZBS12; McNamara and Ford 2000 ) . The transposition activity of ZF-Mos1 was evaluated in E. coli , and was shown to be at least 100-fold lower than the wild-type transposase.…”

Les domaines à doigts de zinc

“…Des études complémentaires ont mis en évidence l'importance de l'acide aminé en position +2 de l'hélice α dans la spécificité de reconnaissance de l'ADN par le motif ZF, en permettant l'interaction avec le brin d'ADN opposé [4]. Cette dernière caractéristique est à prendre en considération lorsque plusieurs motifs ZF sont [5]. Pour construire des ZFP composées de 3 motifs ZF, trois stratégies de sélection sont décrites dans la littérature (Figure 2).…”

“…Les 3 motifs ZF ainsi obtenus ont une affinité très élevée pour la séquence d'ADN choisie, mais cette méthode est longue et difficile d'accès pour de nombreux laboratoires car elle nécessite l'utilisation de plusieurs banques de motifs ZF ( Figure 2B). Enfin, il existe une troisième méthode de sélection combinant les avantages des deux premières, la sélection bipartite [4][5][6][7][8][9][10]. Cette stratégie utilise deux banques, dont chacune contient un doigt de zinc et demi de Zif268.…”