Involvement of phi29 DNA polymerase thumb subdomain in the proper coordination of synthesis and degradation during DNA replication.

Abstract:

Phi29 DNA polymerase achieves a functional coupling between its 3'-5' exonuclease and polymerization activities by means of important contacts with the DNA at both active sites. The placement and orientation of residues Lys538, Lys555, Lys557, Gln560, Thr571, Thr573 and Lys575 in a modelled phi29 DNA polymerase-DNA complex suggest a DNA-binding role. In addition, crystal structure of phi29 DNA polymerase-oligo (dT)5 complex showed Leu567, placed at the tip of the thumb subdomain, lying between the two 3'-terminal bases at the exonuclease site. Single replacement of these phi29 DNA polymerase residues by alanine was made, and mutant derivatives were overproduced and purified to homogeneity. The results obtained in the assay of their synthetic and degradative activities, as well as their coordination, allow us to propose: (1) a primer-terminus stabilization role at the polymerase active site for residues Lys538, Thr573 and Lys575, (2) a primer-terminus stabilization role at the exonuclease active site for residues Leu567 and Lys555 and (3) a primer-terminus binding role in both editing and polymerization modes for residue Gln560. The results presented here lead us to propose phi29 DNA polymerase thumb as the main subdomain responsible for the coordination of polymerization and exonuclease activities.

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