Diabetes Obesity And Metabolism

Conversely, DNA repair enzymes frequently display weaker DNA interactions and incorporate nucleotides more slowly and consequently synthesize DNA in a distributive mode.We used a templateprimer and different enzyme:DNA ratios to study the processivity of human pol. Although it can not be discarded that accessory proteins could modulate processivity, these results suggest that pol is not well suited to carry out long patch DNA synthesis in vivo.We therefore compared the processivity of pols and using a templateprimer and a nucleotide gap with or without a phosphate group at its side. B, the processivity of both enzymes was increased in the presence of a phosphate group at the side of the gap. For pol, this increase was strictly dependent on the presence of a phosphate group, whereas pol showed some increase in processivity. However, this phosphateindependent increase in processivity was only seen when the gap had been partially filled, suggesting the establishment of specific contacts between pol and the DNA requiring a defined gap size. Whereas pol displayed a significant strand displacement capacity and efficiently inserted one additional nucleotide after filling the gap, pol limited its synthesis to the nucleotides of the gap.This imprecise gap filling by pol, which has been already described. C, manganese greatly stimulated strand displacement synthesis on gapped DNA, catalyzed both by pol and pol. For both enzymes, the extension products obtained on the gapped substrate were comparable with those obtained in the control templateprimer molecule, and only a slight increase in the proportion of and products revealed that the reaction was occurring in a gapped molecule.Contrarily, a reduction in nucleotide incorporation was observed when higher amounts. These data suggested that pol was saturated at a low nucleotide concentration.This difference was investigated further using defined DNA molecules and single turnover conditions, where the enzyme concentration is higher than the concentration of DNA.Under these conditions, DNA binding differences between the two enzymes are minimized, and the kinetic constants reflect the equilibrium binding constant. Single turnover analysis of dAMP incorporation clearly revealed that pol has a higher affinity for dAMP than pol. This DNA substrate produces a colorless M plaque phenotype because of a TGA stop codon in the lacZ complementation gene sequence within the gap.As described previously base substitution errors that revert the nonsense codon are scored as blue plaque revertants among total copied and ligated products.DNA sequence analysis of pol generated revertants revealed significant differences in the base substitution specificity of the two enzymes.Polymerase predominantly generates transition errors.Note that of the pol produced base substitutions only were transversion errors.In contrast pol generated base substitutions are more divided evenly between transitions and transversions.Amino acid sequence comparison among all members of this family reveals a common domain organization, which could imply a similar catalytic mechanism. However, despite these general similarities, family X includes heterodox polymerases such as terminal deoxynucleotidyl transferase, able to conduct templateindependent synthesis, and pol, also endowed with some degree of template independence and extremely unfaithful, no evidence had been published to date regarding pol polymerization properties.Moreover, as has been shown recently, pol has an intrinsic dRP lyase activity, an activity that is crucial for the base excision repair pathway.

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