Ferguson ZNF Sandbox: Difference between revisions

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[[Image:2adr.jpg|right|300px]]Two mutants in the N-terminal domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc finger sequences spanning residues 102-159 were studied at the Department of Biochemistry at the University of Washington<ref>Hoffman, R.C.; Horvath, S.J.; Klevit, R.E. Structures of DNA-binding mutant zinc finger domains: implications for DNA binding. Protein Sci. 1993 June; 2(6): 951–965.</ref>. The structure to the left shows the region responsible for DNA binding and contains two zinc fingers. Within this region, there are two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect the DNA-binding activity of ADR1 that been identified: H118A and H118Y. Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which makes a direct DNA contact in the wild-type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed regarding the domain structure and this change can and will affect the affinity of the protein-DNA interaction<ref>Hoffman, R.C.; Horvath, S.J.; Klevit, R.E. Structures of DNA-binding mutant zinc finger domains: implications for DNA binding. Protein Sci. 1993 June; 2(6): 951–965.</ref>.

===Designer Zinc Fingers to Create DNA Scissors===

A breakthrough in the studies of zinc finger proteins has been the ability to cleave sites in a large genome through endonuclease activities<ref>MMMMMM</ref>. FokI endonuclease has two domans; one that binds to DNA and another that cleaves the DNA. By fusing a FokI mononmer with two zinc finger proteins, which bind adjacent sequences, will generate at least an 18 base pair sequence specific DNA nuclease that allows for selective targeting in mammalian genomes<ref>Davis, David, and David Stokoe. "Zinc Finger Nucleases as Tools to Understand and Treat Human Diseases." BMC Medicine 8.1 (2010): 42.</ref>.

As seen in the figure above, two zinc finger trimers are bound to the DNA sequence creating a dimer of FokI. Each zinc finger binds to nine or more nucleotides. After the cleavage by this complex, overhanging ends are left, which sometimes results in deletions or insertions. If this occurs in a coding region, the outcome could be a shift in the reading frame, which can lead to a null allele of the gene that is targeted<ref></ref>.

==References==

Ferguson ZNF Sandbox: Difference between revisions

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@@ Line 28: / Line 28: @@
 [[Image:2adr.jpg|right|300px]]Two mutants in the N-terminal domain of ADR1, a yeast transcription factor that contains two Cys2-His2 zinc finger sequences spanning residues 102-159 were studied at the Department of Biochemistry at the University of Washington<ref>Hoffman, R.C.; Horvath, S.J.; Klevit, R.E. Structures of DNA-binding mutant zinc finger domains: implications for DNA binding. Protein Sci. 1993 June; 2(6): 951–965.</ref>.  The structure to the left shows the region responsible for DNA binding and contains two zinc fingers.  Within this region, there are two point mutants at position 118 in the N-terminal zinc finger (ADR1b: 102-130) that adversely affect the DNA-binding activity of ADR1 that been identified: H118A and H118Y. Comparisons of wild-type ADR1b and the two mutants revealed that neither mutation causes a significant structural perturbation. The structures indicate that the DNA binding properties of the His 118 mutants are dependent on the identity of the side chain at position 118, which makes a direct DNA contact in the wild-type ADR1 protein. The results suggest that the identity of the side chain at the middle DNA contact position in Cys2-His2 zinc fingers may be changed regarding the domain structure and this change can and will affect the affinity of the protein-DNA interaction<ref>Hoffman, R.C.; Horvath, S.J.; Klevit, R.E. Structures of DNA-binding mutant zinc finger domains: implications for DNA binding. Protein Sci. 1993 June; 2(6): 951–965.</ref>.
+===Designer Zinc Fingers to Create DNA Scissors===
+A breakthrough in the studies of zinc finger proteins has been the ability to cleave sites in a large genome through endonuclease activities<ref>MMMMMM</ref>.  FokI endonuclease has two domans; one that binds to DNA and another that cleaves the DNA.  By fusing a FokI mononmer with two zinc finger proteins, which bind adjacent sequences, will generate at least an 18 base pair sequence specific DNA nuclease that allows for selective targeting in mammalian genomes<ref>Davis, David, and David Stokoe. "Zinc Finger Nucleases as Tools to Understand and Treat Human Diseases." BMC Medicine 8.1 (2010): 42.</ref>.
+As seen in the figure above, two zinc finger trimers are bound to the DNA sequence creating a dimer of FokI.  Each zinc finger binds to nine or more nucleotides.  After the cleavage by this complex, overhanging ends are left, which sometimes results in deletions or insertions.  If this occurs in a coding region, the outcome could be a shift in the reading frame, which can lead to a null allele of the gene that is targeted<ref></ref>.
 ==References==
 <references/>