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A pair of ZFNs, each with three zinc fingers binding to target DNA, are shown introducing a double-strand break, at the FokI domain, depicted in yellow. Subsequently, the double strand break is shown as being repaired through either homology-directed repair or non-homologous end joining. [3]
Zinc fingers were first identified in a study of transcription in the African clawed frog, Xenopus laevis in the laboratory of Aaron Klug.A study of the transcription of a particular RNA sequence revealed that the binding strength of a small transcription factor (transcription factor IIIA; TFIIIA) was due to the presence of zinc-coordinating finger-like structures. [6]
The dimerization of two ZFNs is required to produce the necessary double-strand break within the CCR5 gene because the interaction between the FokI enzyme and DNA is weak. [11] This break is repaired by the natural repair mechanisms of the cell, specifically non-homologous end joining. [11]
A double-strand break repair model refers to the various models of pathways that cells undertake to repair double strand-breaks (DSB). DSB repair is an important cellular process, as the accumulation of unrepaired DSB could lead to chromosomal rearrangements, tumorigenesis or even cell death. [ 1 ]
After strand invasion, the further sequence of events may follow either of two main pathways discussed below (see Models); the DSBR (double-strand break repair) pathway or the SDSA (synthesis-dependent strand annealing) pathway. Homologous recombination that occurs during DNA repair tends to result in non-crossover products, in effect restoring ...
Strand exchange then proceeds via a crossed strand intermediate analogous to the Holliday junction in which only one pair of strands has been exchanged. [15] [16] The mechanism and control of serine recombinases is much less well understood. This group of enzymes was only discovered in the mid-1990s and is still relatively small.
MUS81/EME1 is a structure specific endonuclease involved in converting interstrand crosslinks to double-strand breaks in a DNA replication-dependent manner. [12] After introduction of a double-strand break, further steps are required to complete the repair process. If a crosslink is not properly repaired it can block DNA replication. [citation ...
The basic mechanism involved in genetic manipulations through programmable nucleases is the recognition of target genomic loci and binding of effector DNA-binding domain (DBD), double-strand breaks (DSBs) in target DNA by the restriction endonucleases (FokI and Cas), and the repair of DSBs through homology-directed recombination (HDR) or non ...