1l3v: Difference between revisions

Revision as of 14:41, 21 February 2008

Crystal Structure of Bacillus DNA Polymerase I Fragment product complex with 15 base pairs of duplex DNA following addition of dTTP, dATP, dCTP, and dGTP residues.

OverviewOverview

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-A translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a "closed" conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an "open" conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.

About this StructureAbout this Structure

1L3V is a Single protein structure of sequence from Geobacillus stearothermophilus with , and as ligands. Active as DNA-directed DNA polymerase, with EC number 2.7.7.7 Full crystallographic information is available from OCA.

ReferenceReference

Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations., Johnson SJ, Taylor JS, Beese LS, Proc Natl Acad Sci U S A. 2003 Apr 1;100(7):3895-900. Epub 2003 Mar 20. PMID:12649320

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-[[Image:1l3v.jpg|left|200px]]<br /><applet load="1l3v" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1l3v, resolution 1.71&Aring;" />
 '''Crystal Structure of Bacillus DNA Polymerase I Fragment product complex with 15 base pairs of duplex DNA following addition of dTTP, dATP, dCTP, and dGTP residues.'''<br />
 ==Overview==
-DNA polymerases replicate DNA by adding nucleotides to a growing primer, strand while avoiding frameshift and point mutations. Here we present a, series of up to six successive replication events that were obtained by, extension of a primed template directly in a crystal of the thermostable, Bacillus DNA polymerase I. The 6-bp extension involves a 20-A, translocation of the DNA duplex, representing the largest molecular, movement observed in a protein crystal. In addition, we obtained the, structure of a "closed" conformation of the enzyme with a bound, triphosphate juxtaposed to a template and a dideoxy-terminated primer by, constructing a point mutant that destroys a crystal lattice contact, stabilizing the wild-type polymerase in an "open" conformation. Together, these observations allow many of the steps involved in DNA replication to, be observed in the same enzyme at near atomic detail. The successive, replication events observed directly by catalysis in the crystal confirm, the general reaction sequence deduced from observations obtained by using, several other polymerases and further refine critical aspects of the known, reaction mechanism, and also allow us to propose new features that concern, the regulated transfer of the template strand between a preinsertion site, and an insertion site. We propose that such regulated transfer is an, important element in the prevention of frameshift mutations in, high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this, polymerase as a powerful model system for mechanistic studies in which the, structural consequences of mismatches and DNA adducts are observed.
+DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations. Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-A translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a "closed" conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an "open" conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.
 ==About this Structure==
-L3V is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Geobacillus_stearothermophilus Geobacillus stearothermophilus] with SUC, SO4 and MG as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1L3V OCA].
+L3V is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Geobacillus_stearothermophilus Geobacillus stearothermophilus] with <scene name='pdbligand=SUC:'>SUC</scene>, <scene name='pdbligand=SO4:'>SO4</scene> and <scene name='pdbligand=MG:'>MG</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/DNA-directed_DNA_polymerase DNA-directed DNA polymerase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=2.7.7.7 2.7.7.7] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1L3V OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Geobacillus stearothermophilus]]
 [[Category: Single protein]]
-[[Category: Beese, L.S.]]
+[[Category: Beese, L S.]]
-[[Category: Johnson, S.J.]]
+[[Category: Johnson, S J.]]
-[[Category: Taylor, J.S.]]
+[[Category: Taylor, J S.]]
 [[Category: MG]]
 [[Category: SO4]]
@@ Line 25: / Line 25: @@
 [[Category: protein-dna complex]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Tue Nov 20 20:13:53 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 13:41:07 2008''