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Metabolism Mechanism

As expected, exogenous addition of DNA was an absolute requirement.Also, as expected, different specific inhibitors of the eukaryotictype class of DNA polymerases, as phosphonoacetic acid, butylphenyldGTP, butylanilinodATP, and aphidicolin, either only moderately affected or did not inhibit polymerization by ASFV pol X.On the other hand, when the SP oligonucleotide was hybridized to a second oligonucleotide providing a nucleotide template sequence up to six additional nucleotides.This result clearly indicates that DNA polymerization by ASFV pol X is templateprimerdependent.This behavior of ASFV pol X is very similar to those of mammalian polband yeast pol IV, the only known DNA polymerases displaying distributive synthesis under similar reaction conditions.Apparently, these three enzymes dissociate from the DNA after each round of nucleotide addition.Interestingly, ASFV pol X was completely unable to use a templateprimer structure with a mismatch at the primerterminus. It has been observed in other systems that inefficient mismatch extension is mainly due to a slow nucleotide insertion rate rather than to a low affinity for mismatched templateprimer structures. Therefore, the inefficiency in extending a mismatch displayed by ASFV pol X might be interpreted as the requirement for a strict orientation of the primerterminus at the polymerization active site for an efficient catalysis.The insertion efficiency of the different complementary nucleotide of the labeled primer at different nucleotide concentrations.In this sense, a structural basis for metal ion mutagenicity has been recently proposed using polbas a model. When the concentration of dATP or ddATP was progressively increased up to mM, the catalytic efficiency was also linearly increased.However, our approach does not allow a direct estimation of the true nucleotide incorporation rate, since the distributivity of the ASFV pol X is probably the ratelimiting step.Therefore, as expected for a DNA polymerase, ASFV pol X is able to read the position of the nucleotide, preferring deoxynucleotides versus ribonucleotides; moreover, this enzyme, like other members of the pol X family, does not discriminate nucleotides on the basis of a strong selection for the OH group.The presence of the phosphate group did not affect the appearance of stranddisplacement products.As it occurred also during polymerization of a templateprimer structure, ASFV pol X showed no discrimination against the dideoxy form of the complementary substrate. When the reaction was carried out in the presence of T DNA ligase, the mer product obtained with dATP, generating a mer final. This result agrees with the finding that different DNA ligases can substitute for each other in cellfree BER systems and that such backup reactions may also occur in vivo. As a control, lane shows that ASFV pol X is a strict requirement for this repair reaction.Although ASFV genes are usually distant homologs of the corresponding genes from other organisms, sequence similarity studies have proven useful for predicting the functional role of many ASFV proteins.As we characterize here, one of these predicted proteins is a pol btype DNA polymerase most likely involved in viral DNA repair.As the smallest of the known DNA polymerases, ASFV pol X could be envisioned as a good model for the understanding of the structuralfunctional basis of templated nucleotide selection.

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