Chemistry Reference
In-Depth Information
to a cysteine and modified with IOP. While the catalytic group neighbors the DNA in
the specific complex, OP is too far from the substrate in the nonspecific complex, and
thus cannot cleave the DNA. Ultimately, conjugation of OP at position 26 yielded an
artificial nuclease that proceeded to ~90% cleavage with no detectable nonspecific clea-
vage at distal sites.
5.2.5
A Fe-EDTA Artificial Nuclease
An alternative DNA-cleaving agent was used to identify the
subunit DNA contact
sites of E. coli RNA polymerase [39]. Iron-EDTA protein conjugates have previously
been shown to cleave DNA by producing hydroxyl radicals, which ultimately attack
the deoxyribose backbone [40]. To create this artificial nuclease, Minchin and co-work-
ers mutated several residues within the
r
subunit to cysteines. These residues were
conjugated with (S)-1-[p-(bromoacetamido)benzyl]-EDTA (BABE) (Figure 5.5). By
identifying the cleavage products of several related promoters, this Fe-BABE conjugate
confirmed the location of the
r
r
subunit-promoter DNA contact sites.
5.2.6
Concluding Remarks
Through direct conjugation of chemical nucleases to DNA binding proteins, several
groups have successfully created artificial nuclease from proteins with no native nu-
clease ability. These nucleases can utilize the favorable protein-DNA binding ele-
ments and can position the hydrolytic metal close to the DNA backbone for efficient
cleavage [41]. These nucleases have provided useful biological insights and a better
understanding of the molecular details involved in protein-DNA interactions.
Figure 5.5 Procedure for the derivatization of proteins with
(S)-1-[p-(bromoacetamido)benzyl]-EDTA (BABE).
