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J. Biol. Chem., Vol. 265, Issue 24, 14327-14334, Aug, 1990
GP Mullen, EH Serpersu, LJ Ferrin, LA Loeb and AS Mildvan
DNA polymerase I (Pol I) is an enzyme of DNA replication and repair
containing three active sites, each requiring divalent metal ions such as
Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of
water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM)
and approximately 20 weak sites (KD = 600 microM). All bound metal ions
retain one or more water ligands as reflected in enhanced paramagnetic
effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol
I, which lacks the 5',3'-exonuclease domain, retains the tight metal
binding site with little or no change in its affinity for Mn2+, but has
lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of
stoichiometric TMP creates a second tight Mn2+ binding site or tightens a
weak site 100-fold. dGTP together with TMP creates a third tight Mn2+
binding site or tightens a weak site 166-fold. The D424A (the Asp424 to
Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a
weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD
= 3500 microM) in comparison with the wild-type large fragment, and no
effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to
Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-
exonuclease-deficient double mutant has lost the tight metal binding site
and four weak metal binding sites. The binding of dGTP to the polymerase
active site of the D355A,E357A double mutant creates one tight Mn2+ binding
site with a dissociation constant (KD = 3.6 microM), comparable with that
found on the wild-type enzyme, which retains one fast exchanging water
ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded
that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which
is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and
are essential for 3',5'-exonuclease activity, but not for polymerase
activity. Additional weak Mn2+ binding sites are detected on the
3',5'-exonuclease domain, which may be activating, and on the polymerase
domain, which may be inhibitory. The essential divalent metal activator of
the polymerase reaction requires the presence of the dNTP substrate for
tight metal binding indicating that the bound substrate coordinates the
metal.(ABSTRACT TRUNCATED AT 400 WORDS)
Metal binding to DNA polymerase I, its large fragment, and two 3',5'- exonuclease mutants of the large fragment
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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