NORMAL GENOMIC

• Venkatramani R, Radhakrishnan R
• Computational delineation of the catalytic step of a high-fidelity DNApolymerase.
• Protein Sci. 2010; 19: 815-25
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• The Bacillus fragment, belonging to a class of high-fidelity polymerases,demonstrates high processivity (adding approximately 115 bases per DNAbinding event) and exceptional accuracy (1 error in 10(6) nucleotideincorporations) during DNA replication. We present analysis of structuralrearrangements and energetics just before and during the chemical step(phosphodiester bond formation) using a combination of classical moleculardynamics, mixed quantum mechanics molecular mechanics simulations, andfree energy computations. We find that the reaction is associative,proceeding via the two-metal-ion mechanism, and requiring the proton onthe terminal primer O3' to transfer to the pyrophosphate tail of theincoming nucleotide before the formation of the pentacovalent transitionstate. Different protonation states for key active site residues directthe system to alternative pathways of catalysis and we estimate a freeenergy barrier of approximately 12 kcal/mol for the chemical step. Wepropose that the protonation of a highly conserved catalytic aspartic acidresidue is essential for the high processivity demonstrated by the enzymeand suggest that global motions could be part of the reaction free energylandscape.

• Perez-Arnaiz P, Lazaro JM, Salas M, de Vega M
• phi29 DNA polymerase active site: role of residue Val250 as metal-dNTPcomplex ligand and in protein-primed initiation.
• J Mol Biol. 2010; 395: 223-33
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• DNA polymerases require two acidic residues to coordinate metal ions A andB at their polymerisation active site during catalysis of nucleotideincorporation. Crystallographic resolution of varphi29 DNA polymeraseternary complex showed that metal B coordination also depends on thecarbonyl group of Val250 that belongs to the highly conserved Dx(2)SLYPmotif of eukaryotic-type (family B) DNA polymerases. In addition, multiplesequence alignments have shown the specific conservation of this residueamong the DNA polymerases that use a protein as primer. Thus, to ascertainits role in polymerisation, we have analysed the behaviour of singlemutations introduced at the corresponding Val250 of varphi29 DNApolymerase. The differences in nucleotide binding affinity shown bymutants V250A and V250F with respect to the wild-type DNA polymerase agreeto a role for Val250 as a metal B-dNTP complex ligand. In addition, mutantV250F was severely affected in varphi29 DNA replication because of a largereduction in the catalytic efficiency of the protein-primed reactions. Inthe light of the varphi29 DNA polymerase structures, a role for Val250residue in the maintenance of the proper architecture of the enzyme toperform the protein-primed reactions is also proposed.

• Banos B, Lazaro JM, Villar L, Salas M, de Vega M
• Editing of misaligned 3'-termini by an intrinsic 3'-5' exonucleaseactivity residing in the PHP domain of a family X DNA polymerase.
• Nucleic Acids Res. 2008; 36: 5736-49
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• Bacillus subtilis gene yshC encodes a family X DNA polymerase (PolX(Bs)),whose biochemical features suggest that it plays a role during DNA repairprocesses. Here, we show that, in addition to the polymerization activity,PolX(Bs) possesses an intrinsic 3'-5' exonuclease activity specialized inresecting unannealed 3'-termini in a gapped DNA substrate. Biochemicalanalysis of a PolX(Bs) deletion mutant lacking the C-terminal polymerasehistidinol phosphatase (PHP) domain, present in most of thebacterial/archaeal PolXs, as well as of this separately expressed proteinregion, allow us to state that the 3'-5' exonuclease activity of PolX(Bs)resides in its PHP domain. Furthermore, site-directed mutagenesis ofPolX(Bs) His339 and His341 residues, evolutionary conserved in the PHPsuperfamily members, demonstrated that the predicted metal binding site isdirectly involved in catalysis of the exonucleolytic reaction. Theimplications of the unannealed 3'-termini resection by the 3'-5'exonuclease activity of PolX(Bs) in the DNA repair context are discussed.

• Parker EJ, Botting CH, Webster A, Hay RT
• Adenovirus DNA polymerase: domain organisation and interaction withpreterminal protein.
• Nucleic Acids Res. 1998; 26: 1240-7
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• Adenovirus DNA polymerase is one of three viral proteins and two cellularproteins required for replication of the adenovirus genome. Duringinitiation of viral DNA synthesis the viral DNA polymerase transfers dCMPonto the adenovirus preterminal protein, to which it is tightly bound. Thedomain structure of the 140 kDa DNA polymerase has been probed by partialproteolysis and the sites of proteolytic cleavage determined by N-terminalsequencing. At least four domains can be recognised within the DNApolymerase. Adenovirus preterminal protein interacts with three of thefour proteolytically derived domains. This was confirmed by cloning andexpression of each of the individual domains. These data indicate that,like other members of the pol alpha family of DNA polymerases, theadenovirus DNA polymerase has a multidomain structure and that interactionwith preterminal protein takes place with non-contiguous regions of thepolypeptide chain over a large surface area of the viral DNA polymerase.

• Koonin EV, Deutscher MP
• RNase T shares conserved sequence motifs with DNA proofreadingexonucleases.
• Nucleic Acids Res. 1993; 21: 2521-2

• Blasco MA, Bernad A, Blanco L, Salas M
• Characterization and mapping of the pyrophosphorolytic activity of thephage phi 29 DNA polymerase. Involvement of amino acid motifs highlyconserved in alpha-like DNA polymerases.
• J Biol Chem. 1991; 266: 7904-9
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• The phi 29 DNA polymerase, an alpha-like DNA polymerase, shows aninorganic pyrophosphate-dependent degradative activity with similarrequirements to the corresponding one of Escherichia coli DNA polymeraseI: (a) it requires a high concentration of inorganic pyrophosphate and isreversed by polymerization; (b) like DNA polymerization, it needs a duplexDNA with protruding 5' single-strand; (c) it acts in the 3' to 5'direction releasing free dNTPs, thus, it can be considered as the reversalof polymerization; (d) although a correctly base-paired 3' primer terminusis the preferred substrate, the pyrophosphorolytic activity is able toremove mismatched 3' ends. In agreement with the structural and functionalmodel previously proposed for the phi 29 DNA polymerase, the analysis ofpoint mutations has revealed that the pyrophosphorolytic activity, likethe polymerization activity, is located at the C-terminal portion of themolecule, involving the amino acid motif YCDTD, highly conserved inalpha-like DNA polymerases. Furthermore, the analysis of phi 29 DNApolymerase mutants indicates that pyrophosphorolysis, like DNApolymerization, also requires an efficient translocation of the enzymealong the template.

• Larder BA, Kemp SD, Darby G
• Related functional domains in virus DNA polymerases.
• EMBO J. 1987; 6: 169-75
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• Analysis of the lesions in several drug-resistant DNA polymerase mutantsof herpes simplex virus along with comparative analysis of the publishedpolymerase sequences of other human herpesviruses has shown that mostlesions (five out of six) are substitutions at amino acid residuesconserved in all four polymerases. Furthermore, the majority of lesionsare in regions of the polypeptide where there are marked clusterings ofconserved residues. On the basis of these data we have identified severaldomains within the polypeptide which we believe may have importantfunctional roles in the action of the enzyme. The apparent restriction inthe potential sites of lesions conferring drug resistance may explain thedifficulty in selecting such mutants using acyclovir (ACV) in culture andtheir failure to emerge so far during ACV therapy. Extension of thecomparative analysis to the polymerases of adenovirus type 2, vacciniavirus and phage phi 29 suggests that these enzymes also possess domainshomologous to those most conserved in the herpes polymerases (regionsI-III) and that these domains have a similar linear spatial distributionon the polypeptides. The results are discussed in relation to the knownfunction of the DNA polymerases.