NORMAL GENOMIC

• Perona JJ, Oza JP
• Crystal structure of a reverse polymerase.
• Proc Natl Acad Sci U S A. 2010; 107: 20149-50

• Croteau DL, Peng Y, Van Houten B
• DNA repair gets physical: mapping an XPA-binding site on ERCC1.
• DNA Repair (Amst). 2008; 7: 819-26
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• Two recent reports provide new physical information on how the XPA proteinrecruits the ERCC1-XPF heterodimer to the site of damage during theprocess of mammalian nucleotide excision repair (NER). Using chemicalshift perturbation NMR experiments, the contact sites between a centralfragment of ERCC1 and an XPA fragment have been mapped. While both studiesagree with regard to the XPA-binding site, they differ on whether theERCC1-XPA complex can simultaneously bind DNA. These studies haveimportant implications for both the molecular process and the design ofpotential inhibitors of NER.

• 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.

• Chen LT, Ko TP, Chang YW, Lin KA, Wang AH, Wang TF
• Structural and functional analyses of five conserved positively chargedresidues in the L1 and N-terminal DNA binding motifs of archaeal RADAprotein.
• PLoS One. 2007; 2: 858-858
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• RecA family proteins engage in an ATP-dependent DNA strand exchangereaction that includes a ssDNA nucleoprotein helical filament and ahomologous dsDNA sequence. In spite of more than 20 years of efforts, themolecular mechanism of homology pairing and strand exchange is still notfully understood. Here we report a crystal structure of Sulfolobussolfataricus RadA overwound right-handed filament with three monomers perhelical pitch. This structure reveals conformational details of the firstssDNA binding disordered loop (denoted L1 motif) and the dsDNA bindingN-terminal domain (NTD). L1 and NTD together form an outwardly open palmstructure on the outer surface of the helical filament. Inside this palmstructure, five conserved basic amino acid residues (K27, K60, R117, R223and R229) surround a 25 A pocket that is wide enough to accommodateanionic ssDNA, dsDNA or both. Biochemical analyses demonstrate that thesefive positively charged residues are essential for DNA binding and forRadA-catalyzed D-loop formation. We suggest that the overwoundright-handed RadA filament represents a functional conformation in thehomology search and pairing reaction. A new structural model is proposedfor the homologous interactions between a RadA-ssDNA nucleoproteinfilament and its dsDNA target.

• Doddridge ZA, Bertram RD, Hayes CJ, Soultanas P
• Effects of vinylphosphonate internucleotide linkages on the cleavagespecificity of exonuclease III and on the activity of DNA polymerase I.
• Biochemistry. 2003; 42: 3239-46
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• We have previously reported the synthesis of vinylphosphonate-linkedthymidine dimers and their incorporation into synthetic oligonucleotidesto create vinylphosphonate internucleotide linkages in the DNA. Suchlinkages have a profound effect on DNA backbone rotational flexibility,and we have shown that the PcrA helicase, which requires such flexibility,is inhibited when it encounters these linkages on the translocatingstrand. In this study, we have investigated the effects of these linkageson the dsDNA specific exonuclease III and on the ssDNA specific mung beannuclease to establish whether our modification confers resistance tonucleases making it suitable for antisense therapy applications. We alsoinvestigated the effect on DNA polymerase I to establish whether we couldin the future use this enzyme to incorporate these linkages in the DNA.Our results show that a single modification does not affect the activityof DNA polymerase I, but four vinylphosphonate linkages in tandem inhibitits activity. Furthermore, such linkages do not confer significantnuclease resistance to either exonuclease III or mung bean nuclease, butunexpectedly, they alter the cleavage specificity of exonuclease III.

• Kawakami J, Kamiya H, Yasuda K, Fujiki H, Kasai H, Sugimoto N
• Thermodynamic stability of base pairs between 2-hydroxyadenine andincoming nucleotides as a determinant of nucleotide incorporationspecificity during replication.
• Nucleic Acids Res. 2001; 29: 3289-96
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• We investigated the thermodynamic stability of double-stranded DNAs withan oxidative DNA lesion, 2-hydroxyadenine (2-OH-Ade), in two differentsequence contexts (5'-GA*C-3' and 5'-TA*A-3', A* represents 2-OH-Ade).When an A*-N pair (N, any nucleotide base) was located in the center of aduplex, the thermodynamic stabilities of the duplexes were similar for allthe natural bases except A (N = T, C and G). On the other hand, for theduplexes with the A*-N pair at the end, which mimic the nucleotideincorporation step, the stabilities of the duplexes were dependent ontheir sequence. The order of stability is T > G > C >> A in the 5'-GA*C-3'sequences and T > A > C > G in the 5'-TA*A-3' sequences. Because T/G/C andT/A are nucleotides incorporated opposite to 2-OH-Ade in the 5'-GA*C-3'and 5'-TA*A-3' sequences, respectively, these results agree with thetendency of mutagenic misincorporation of the nucleotides opposite to2-OH-Ade in vitro. Thus, the thermodynamic stability of the A*-N base pairmay be an important factor for the mutation spectra of 2-OH-Ade.

• Morales JC, Kool ET
• Minor Groove Interactions between Polymerase and DNA: More Essential toReplication than Watson-Crick Hydrogen Bonds?
• J Am Chem Soc. 1999; 121: 2323-2324

• Bebenek K, Boyer JC, Kunkel TA
• The base substitution fidelity of HIV-1 reverse transcriptase on DNA andRNA templates probed with 8-oxo-deoxyguanosine triphosphate.
• Mutat Res. 1999; 429: 149-58
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• We have used 8-O-dGTP, a mutagenic nucleotide generated by oxidativemetabolism, to probe the misincorporation potential of HIV-1 reversetranscriptase (RT) during DNA synthesis templated by the same nucleotidesequence as either RNA or DNA. With either template, 8-O-dGMP wasmisincorporated opposite template A, yielding characteristic A-->Ctransversions. The error rate with DNA was similar to that with RNA,suggesting that base misincorporation by the RT during first-strand andsecond-strand replication may contribute equally to the HIV-1 basesubstitution mutation rate. The rate of 8-O-dGMP misincorporation differedby more than 10-fold among the 20 adenines in the M13mp2 template whereA-->C transversions can be detected. The transversion distribution wassimilar with the two templates, indicating that the effects of flankingnucleotides on misincorporation rates were similar. This is consistentwith structural and biochemical data suggesting that HIV-1 RT binds RNA xDNA and DNA x DNA template-primers in the same orientation. Thesimilarities in error rates and distribution further indicate that,despite differences in the structures of free RNA x DNA and DNA x DNAduplexes (e.g., minor groove dimensions), the polymerase active site thatassembles upon substrate binding establishes a similar degree ofnucleotide selectivity with both types of template-primers. Comparison ofthe RT error distribution to that observed with two Pol I family DNApolymerases and a Pol alpha family polymerase revealed common hot and coldspots for misincorporation. This suggests that the local nucleotidesequence influences the nucleotide selectivity of four polymerases in asimilar manner, despite their differences in structure, biochemicalproperties, and functions.

• DiGiuseppe JA, Wright GE, Dresler SL
• A kinetic study of rat recombinant DNA polymerase beta: detection of aslow (hysteretic) transition in polymerase activity and inhibition bybutylphenyl-deoxyguanosine triphosphate.
• Nucleic Acids Res. 1989; 17: 3079-89
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• We have identified and characterized a distinct non-linearity in the timecourse of the reaction of mammalian DNA polymerase beta with syntheticpolynucleotides. Nucleotide incorporation is biphasic; an initial burst ofactivity decays exponentially to a lower steady-state velocity. This slowtransition in polymerase activity is not due to substrate depletion,abortive complex formation, or enzyme inactivation. The data areconsistent with description of the beta-polymerase as a hysteretic enzyme,a finding which provides a potential explanation for the non-hyperbolickinetics which have been reported previously for this polymerase. We havealso found, in contrast to some previous data, that the nucleotideanalogue, N2-(p-n-butylphenyl)-2'-deoxyguanosine-5'-triphosphate(BuPdGTP), is an inhibitor of the beta-polymerase. When poly(dC).oligo(dG)is used as template.primer, inhibition of the initial velocity iscompetitive with dGTP with a Ki of 1.25 microM. On activated DNA, however,beta-polymerase displays sensitivity to BuPdGTP which overlaps with thatpreviously reported for DNA polymerase delta; 100 microM BuPdGTP isrequired to inhibit the initial velocity of a dGTP-deficient, truncatedassay. Finally, we demonstrate that, in addition to its inhibition ofinitial velocity, BuPdGTP also modulates both the rate constant of theslow transition in polymerase activity, and the steady-state velocity ofthe beta-polymerase.

• Rozovskaia TA, Chenchik AA, Tarusova NB, Bibilashvili RSh, Khomutov RM
• [Pyrophosphate analogs in the pyrophosphorolysis reaction catalyzed byEscherichia coli RNA polymerase].
• Mol Biol (Mosk). 1981; 15: 1205-23
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• Processive pyrophosphorolysis of RNA from ternary RNA polymerase-nascentRNA-delta D111 T7 DNA complex has been followed in the absence ofnucleoside triphosphates. Series of inorganic pyrophosphate analogs wereinvestigated for their ability to sustain the reaction and to compete withinorganic pyrophosphate for the reaction. Methylenediphosphonic,imidodiphosphonic, phosphonacetic acids, inorganic triphosphate,methylenediphosphonic and phosphate were found to be capable ofsubstituting the inorganic pyrophosphate in RNA degradation reaction withtantamount efficiency. They give rise to nucleoside monophosphates forphosphonoacetic acid, nucleoside triphosphates for inorganic pyrophosphateand inorganic triphosphate, nucleoside triphosphates analogs formethylenediphosphonic, imidodiphosphonic acids and methylenediphosphonicacid phosphate as the low molecular weight product of the reaction. Theproblem of specific interaction of RNA polymerase with nucleosidetriphosphates and inorganic pyrophosphate is discussed in the terms ofstructural requirements for the compounds to be a potent substrate for RNApolymerase.

• Kerrich-Santo RE, Hartmann GR
• Influence of temperature on the action of rifampicin on RNA polymerase inpresence of DNA.
• Eur J Biochem. 1974; 43: 521-32