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| - | [[Image:7ice.gif|left|200px]]<br /> | |
| - | <applet load="7ice" size="450" color="white" frame="true" align="right" spinBox="true" | |
| - | caption="7ice, resolution 2.800Å" /> | |
| - | '''DNA POLYMERASE BETA (E.C.2.7.7.7)/DNA COMPLEX, SOAKED IN THE PRESENCE OF CACL2'''<br /> | |
| | | | |
| - | ==Overview== | + | ==DNA POLYMERASE BETA (E.C.2.7.7.7)/DNA COMPLEX, SOAKED IN THE PRESENCE OF CACL2== |
| - | Mammalian DNA polymerase beta (pol beta) is a small (39 kDa) DNA, gap-filling enzyme that comprises an amino-terminal 8-kDa domain and a, carboxy-terminal 31-kDa domain. In the work reported here, crystal, structures of human pol beta complexed with blunt-ended segments of DNA, show that, although the crystals belong to a different space group, the, DNA is nevertheless bound in the pol beta binding channel in the same way, as the DNA in previously reported structures of rat pol beta complexed, with a template-primer and ddCTP [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., & Kraut, J. (1994) Science 264, 1891-1903]. The 8-kDa, domain is in one of three previously observed positions relative to the, 31-kDa domain, suggesting that the 8-kDa domain may assume only a small, number of stable conformations. The thumb subdomain is in a more open, position in the human pol beta-DNA binary complex than it is in the rat, pol beta-DNA-ddCTP ternary complex, and a closing thumb upon nucleotide, binding could represent the rate-limiting conformational change that has, been observed in pre-steady-state kinetic studies. Intermolecular contacts, between the DNA and the 8-kDa domain of a symmetry-related pol beta, molecule reveal a plausible binding site on the 8-kDa domain for the, downstream oligonucleotide of a gapped-DNA substrate; in addition to a, lysine-rich binding pocket that accommodates a 5'-PO4 end group, the 8-kDa, domain also contains a newly discovered helix-hairpin-helix (HhH) motif, that binds to DNA in the same way as does a structurally and sequentially, homologous HhH motif in the 31-kDa domain. DNA binding by both HhH motifs, is facilitated by a metal ion. In that HhH motifs have been identified in, other DNA repair enzymes and DNA polymerases, the HhH-DNA interactions, observed in pol beta may be applicable to a broad range of DNA binding, proteins. The sequence similarity between the HhH motif of endonuclease, III from Escherichia coli and the HhH motif of the 8-kDa domain of pol, beta is particularly striking in that all of the conserved residues are, clustered in one short sequence segment, LPGVGXK, where LPGV corresponds, to a type II beta-turn (the hairpin turn), and GXK corresponds to a part, of the HhH motif that is proposed to be critical for DNA binding and, catalysis for both enzymes. These results suggest that endonuclease III, and the 8-kDa domain of pol beta may employ a similar mode of DNA binding, and may have similar catalytic mechanisms for their respective DNA lyase, activities. A model for productive binding of pol beta to a gapped-DNA, substrate requires a 90 degrees bend in the single-stranded template, which could enhance nucleotide selectivity during DNA repair or, replication.
| + | <StructureSection load='7ice' size='340' side='right'caption='[[7ice]], [[Resolution|resolution]] 2.80Å' scene=''> |
| | + | == Structural highlights == |
| | + | <table><tr><td colspan='2'>[[7ice]] is a 3 chain structure with sequence from [https://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=7ICE OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=7ICE FirstGlance]. <br> |
| | + | </td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 2.8Å</td></tr> |
| | + | <tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=NA:SODIUM+ION'>NA</scene></td></tr> |
| | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=7ice FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=7ice OCA], [https://pdbe.org/7ice PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=7ice RCSB], [https://www.ebi.ac.uk/pdbsum/7ice PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=7ice ProSAT]</span></td></tr> |
| | + | </table> |
| | + | == Function == |
| | + | [https://www.uniprot.org/uniprot/DPOLB_HUMAN DPOLB_HUMAN] Repair polymerase that plays a key role in base-excision repair. Has 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity that removes the 5' sugar phosphate and also acts as a DNA polymerase that adds one nucleotide to the 3' end of the arising single-nucleotide gap. Conducts 'gap-filling' DNA synthesis in a stepwise distributive fashion rather than in a processive fashion as for other DNA polymerases.<ref>PMID:9207062</ref> <ref>PMID:9572863</ref> <ref>PMID:11805079</ref> <ref>PMID:21362556</ref> |
| | + | == Evolutionary Conservation == |
| | + | [[Image:Consurf_key_small.gif|200px|right]] |
| | + | Check<jmol> |
| | + | <jmolCheckbox> |
| | + | <scriptWhenChecked>; select protein; define ~consurf_to_do selected; consurf_initial_scene = true; script "/wiki/ConSurf/ic/7ice_consurf.spt"</scriptWhenChecked> |
| | + | <scriptWhenUnchecked>script /wiki/extensions/Proteopedia/spt/initialview01.spt</scriptWhenUnchecked> |
| | + | <text>to colour the structure by Evolutionary Conservation</text> |
| | + | </jmolCheckbox> |
| | + | </jmol>, as determined by [http://consurfdb.tau.ac.il/ ConSurfDB]. You may read the [[Conservation%2C_Evolutionary|explanation]] of the method and the full data available from [http://bental.tau.ac.il/new_ConSurfDB/main_output.php?pdb_ID=7ice ConSurf]. |
| | + | <div style="clear:both"></div> |
| | + | <div style="background-color:#fffaf0;"> |
| | + | == Publication Abstract from PubMed == |
| | + | When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template. |
| | | | |
| - | ==About this Structure==
| + | A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta.,Pelletier H, Sawaya MR, Wolfle W, Wilson SH, Kraut J Biochemistry. 1996 Oct 1;35(39):12762-77. PMID:8841119<ref>PMID:8841119</ref> |
| - | 7ICE is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Homo_sapiens Homo sapiens] with NA as [http://en.wikipedia.org/wiki/ligand ligand]. 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=7ICE OCA].
| + | |
| | | | |
| - | ==Reference==
| + | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
| - | Crystal structures of human DNA polymerase beta complexed with DNA: implications for catalytic mechanism, processivity, and fidelity., Pelletier H, Sawaya MR, Wolfle W, Wilson SH, Kraut J, Biochemistry. 1996 Oct 1;35(39):12742-61. PMID:[http://ispc.weizmann.ac.il//pmbin/getpm?pmid=8841118 8841118]
| + | </div> |
| - | [[Category: DNA-directed DNA polymerase]]
| + | <div class="pdbe-citations 7ice" style="background-color:#fffaf0;"></div> |
| - | [[Category: Homo sapiens]]
| + | |
| - | [[Category: Single protein]]
| + | |
| - | [[Category: Pelletier, H.]]
| + | |
| - | [[Category: Sawaya, M.R.]]
| + | |
| - | [[Category: NA]]
| + | |
| - | [[Category: complex (nucleotidyltransferase/dna]]
| + | |
| - | [[Category: dna repair]]
| + | |
| - | [[Category: dna replication]]
| + | |
| - | [[Category: dna-directed dna polymerase]]
| + | |
| - | [[Category: nucleotidyltransferase]]
| + | |
| | | | |
| - | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 12 23:53:18 2007''
| + | ==See Also== |
| | + | *[[DNA polymerase 3D structures|DNA polymerase 3D structures]] |
| | + | == References == |
| | + | <references/> |
| | + | __TOC__ |
| | + | </StructureSection> |
| | + | [[Category: Homo sapiens]] |
| | + | [[Category: Large Structures]] |
| | + | [[Category: Pelletier H]] |
| | + | [[Category: Sawaya MR]] |
| Structural highlights
Function
DPOLB_HUMAN Repair polymerase that plays a key role in base-excision repair. Has 5'-deoxyribose-5-phosphate lyase (dRP lyase) activity that removes the 5' sugar phosphate and also acts as a DNA polymerase that adds one nucleotide to the 3' end of the arising single-nucleotide gap. Conducts 'gap-filling' DNA synthesis in a stepwise distributive fashion rather than in a processive fashion as for other DNA polymerases.[1] [2] [3] [4]
Evolutionary Conservation
Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf.
Publication Abstract from PubMed
When crystals of human DNA polymerase beta (pol beta) complexed with DNA [Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742-12761] are soaked in the presence of dATP and Mn2+, X-ray structural analysis shows that nucleotidyl transfer to the primer 3'-OH takes place directly in the crystals, even though the DNA is blunt-ended at the active site. Under similar crystal-soaking conditions, there is no evidence for a reaction when Mn2+ is replaced by Mg2+, which is thought to be the divalent metal ion utilized by most polymerases in vivo. These results suggest that one way Mn2+ may manifest its mutagenic effect on polymerases is by promoting greater reactivity than Mg2+ at the catalytic site, thereby allowing the nucleotidyl transfer reaction to take place with little or no regard to instructions from a template. Non-template-directed nucleotidyl transfer is also observed when pol beta-DNA cocrystals are soaked in the presence of dATP and Zn2+, but the reaction products differ in that the sugar moiety of the incorporated nucleotide appears distorted or otherwise cleaved, in agreement with reports that Zn2+ may act as a polymerase inhibitor rather than as a mutagen [Sirover, M. A., & Loeb, L. A. (1976) Science 194, 1434-1436]. Although no reaction is observed when crystals are soaked in the presence of dATP and other metal ions such as Ca2+, Co2+, Cr3+, or Ni2+, X-ray structural analyses show that these metal ions coordinate the triphosphate moiety of the nucleotide in a manner that differs from that observed with Mg2+. In addition, all metal ions tested, with the exception of Mg2+, promote a change in the side-chain position of aspartic acid 192, which is one of three highly conserved active-site carboxylate residues. Soaking experiments with nucleotides other than dATP (namely, dCTP, dGTP, dTTP, ATP, ddATP, ddCTP, AZT-TP, and dATP alpha S) reveal a non-base-specific binding site on pol beta for the triphosphate and sugar moieties of a nucleotide, suggesting a possible mechanism for nucleotide selectivity whereby triphosphate-sugar binding precedes a check for correct base pairing with the template.
A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta.,Pelletier H, Sawaya MR, Wolfle W, Wilson SH, Kraut J Biochemistry. 1996 Oct 1;35(39):12762-77. PMID:8841119[5]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
See Also
References
- ↑ Bennett RA, Wilson DM 3rd, Wong D, Demple B. Interaction of human apurinic endonuclease and DNA polymerase beta in the base excision repair pathway. Proc Natl Acad Sci U S A. 1997 Jul 8;94(14):7166-9. PMID:9207062
- ↑ Matsumoto Y, Kim K, Katz DS, Feng JA. Catalytic center of DNA polymerase beta for excision of deoxyribose phosphate groups. Biochemistry. 1998 May 5;37(18):6456-64. PMID:9572863 doi:10.1021/bi9727545
- ↑ DeMott MS, Beyret E, Wong D, Bales BC, Hwang JT, Greenberg MM, Demple B. Covalent trapping of human DNA polymerase beta by the oxidative DNA lesion 2-deoxyribonolactone. J Biol Chem. 2002 Mar 8;277(10):7637-40. Epub 2002 Jan 22. PMID:11805079 doi:10.1074/jbc.C100577200
- ↑ Parsons JL, Dianova II, Khoronenkova SV, Edelmann MJ, Kessler BM, Dianov GL. USP47 is a deubiquitylating enzyme that regulates base excision repair by controlling steady-state levels of DNA polymerase beta. Mol Cell. 2011 Mar 4;41(5):609-15. doi: 10.1016/j.molcel.2011.02.016. PMID:21362556 doi:10.1016/j.molcel.2011.02.016
- ↑ Pelletier H, Sawaya MR, Wolfle W, Wilson SH, Kraut J. A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta. Biochemistry. 1996 Oct 1;35(39):12762-77. PMID:8841119 doi:http://dx.doi.org/10.1021/bi9529566
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