[“Metabolism Worksheet

Polymerization activity was calculated as the amount of incorporated dNMP.After incubation for different times at C, the reactions were stopped by adding EDTA up to mM.Samples were analyzed by M urea, PAGE and autoradiography.Exonuclease activity would be detected as a decrease in the size of the labeled SP primer.Samples were analyzed by M urea, PAGE and autoradiography.Samples were analyzed by M urea, PAGE and autoradiography.Quantitation was done by densitometric analysis of the band corresponding to primer extension products.Nucleotide insertion on each hybrid structure was comparatively studied as described above, by providing ng of ASFV pol X and various concentrations of either the correct dNTP. When indicated, T DNA ligase was added to the reaction mixture to seal the repaired DNA gap.The regions corresponding to some stretches of secondary structure elements of both palm and thumb subdomains could be aligned without significant gaps among the different sequences, including ASFV pol X.This conservation at the primary structure level is probably the reflect of superior order constraints to form a conserved catalytic core.Mitochondrial polbdiffers from cellular pol bs in larger intervening sequences connecting secondary structure elements, particularly that between bstrand and ahelix O.Contrarily, and contributing to its reduced size, ASFV pol X lacks some intervening amino acid sequences, such as that among bstrand and ahelix L, which is particularly extensive in the case of yeast pol IV.In support of its functional significance, the inclusion of ASFV pol X in this multiple alignment outlines a very limited number, that include the catalytic triad of aspartates involved in metal binding. Interestingly, the thumb is the subdomain showing the highest similarity among the sequences compared, containing eight invariant residues.On the contrary, the palm subdomain, that contains seven invariant residues, including the three critical aspartates, allows larger differences in the number of residues connecting several secondary structure elements.strongly indicates an evolutionary relationship between ASFV pol X and DNA polymerases from family X.Numbers between slashes indicate the amino acid position relative to the N terminus of each DNA polymerase.Anarrow shows the expected position for protein pOL. The DNA polymerase activity band corresponding to the electrophoretic migration of protein pOL is indicated as ASFV pol X.Arrows indicate the sedimentation position of several molecular mass markers centrifuged in control gradients.According to rat polbstructural data, fingers, and thumb are indicated at the top of the aligned sequences.The portions of the alignment that correspond to these secondary structure elements are included in blue areas.Two helixhairpinhelix motifs are between ahelices C and D. Residues of the palm and thumb subdomains that are invariant among all the sequences compared and with an asterisk at the top of the alignment.Any other identities with ASFV pol X in these two subdomains are in boldface type.Colored dots at the bottom of the alignment indicate polbresidues, dNTP ligands, and metal ligands, which are invariant or highly conserved in ASFV pol X and other pol X members.Light blue dots indicate polbresidues involved in interactions between the palm and thumb subdomains. Numbers in parentheses indicate the total number of amino acid residues of each DNA polymerase.

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