Methods in Molecular Biology • 16 Enzymes of Molecular Biology

(Nancy Kaufman) #1
BAL 31 Nucleases 237

single- and double-stranded substrates in the presence of 5% SDS if
both Mg 2÷ and Ca 2÷ were present prior to exposure to the detergent (3).
Preliminary experiments on the S enzyme showed that substantial
activity on single-stranded DNA was observed in up to 6M guanidinium
hydrochloride (5).
Cleavage of adducts of nonsupercoiled closed duplex DNA with
Hg 2÷ or Ag ÷ by the F nuclease (13, 2 7) suggested that the enzyme is not
highly sensitive to these metal ions, but it must be pointed out that, in
the case of Ag ÷, virtually all the metal ion was bound to DNA nucle-
otide. The only metal ion tested that appeared to be strongly inhibitory
was Zn 2÷ (28). Inhibitors of unknown nature that can very strongly
repress the duplex exonuclease activity of the BAL 31 nucleases have
been observed in preparations of plasmid DNA prepared by rapid
procedures, such as the alkaline lysis protocol of Birnboim and Doly
(29), that do not involve purification by centrifugation to equilibrium
in CsC1 density gradients containing intercalating compounds such as
ethidium bromide (30,31). An additional purification step, using small
columns of Bio-Gel A-0.5m agarose-based size exclusion resin (Bio-
Rad Laboratories, Richmond, CA), has been shown to render the DNA
digestible by commercially provided (Gibco BRL, Grand Island, NY)
BAL 31 nuclease, thus avoiding the time-consuming equilibrium ultra-
centrifugation procedure (30).
Another unexplained strong source of inhibition was found as a com-
ponent of a T4 DNA polymerase repair reaction: DNA that had been thus
treated, and the polymerase inactivated, without subsequent precipitation
and resuspension of the DNA was virtually unaffected by the duplex
exonuclease activity (5). The inhibitor was shown not to be the 5'-deoxy-
nucleoside triphosphate substrates for the polymerase and was not a
large macromole since the nuclease activity on the repaired DNA was
restored when it was passed through a Sephadex ® G25 "spun column."
The 5'-deoxynucleoside monophosphates (5'-dNMPs) resulting from
nuclease action (4) are apparently inhibitory because the velocity of
the duplex exonuclease reaction decreases significantly with time,
even though the substrate concentration (concentration of DNA ends)
remains constant until molecules begin to be completely degraded (6).
The nuclease is known to bind to 5'-dNMPs, because an affinity col-
umn consisting of 5'-dNMPs covalently attached to agarose is used in
the purification procedure (4).

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