Processive DNA synthesis observed in a polymerase crystal suggests a mechanism for the prevention of frameshift mutations

Abstract

DNA polymerases replicate DNA by adding nucleotides to a growing primer strand while avoiding frameshift and point mutations.Here we present a series of up to six successive replication events that were obtained by extension of a primed template directly in a crystal of the thermostable Bacillus DNA polymerase I. The 6-bp extension involves a 20-Å translocation of the DNA duplex, representing the largest molecular movement observed in a protein crystal. In addition, we obtained the structure of a ''closed'' conformation of the enzyme with a bound triphosphate juxtaposed to a template and a dideoxy-terminated primer by constructing a point mutant that destroys a crystal lattice contact stabilizing the wild-type polymerase in an ''open'' conformation. Together, these observations allow many of the steps involved in DNA replication to be observed in the same enzyme at near atomic detail. The successive replication events observed directly by catalysis in the crystal confirm the general reaction sequence deduced from observations obtained by using several other polymerases and further refine critical aspects of the known reaction mechanism, and also allow us to propose new features that concern the regulated transfer of the template strand between a preinsertion site and an insertion site. We propose that such regulated transfer is an important element in the prevention of frameshift mutations in high-fidelity DNA polymerases. The ability to observe processive, high-fidelity replication directly in a crystal establishes this polymerase as a powerful model system for mechanistic studies in which the structural consequences of mismatches and DNA adducts are observed.

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

“…Moreover, structures have been identified that reveal the different stages in the replication cycle ( Figure 1a) and highlight the conformational transitions of the polymerase during this process [11].…”

“…In the ternary complex -containing dNTP and DNA (Figure 1a) -the Tyr714 residue is displaced from the insertion site to a position behind the nascent base pair, thus enabling pairing of the incoming dNTP and the templating (n) base. BF is in a 'closed' conformation, resulting from a ~40° rotation of the O helix from its 'open' position [11]. Specifically, the O helix of the fingers domain closes down towards the flat surface of the nascent base pair, resulting in a tight-fitting, reaction-ready ternary complex.…”

“…Aside from translesion synthesis, it has recently been shown that at least some Y-family DNA polymerases are involved in other functions, including recombination and somatic hypermutation [8,9,66]. BF features a sterically tight active site and an exonuclease domain {non-functional in the strain used in crystallographic studies [11,31,62] but functional in Bst 320 ATCC55719 (http://www.patentstorm.us/patents/5747298-claims.html)}, but lacks a little finger domain, whereas Dpo4 lacks an exonuclease domain but has an additional little finger domain, and a sterically more open active site. Cartoon representations of BF (a) and Dpo4 (b).…”

“…The minor-groove side of this base pair indicated by the red arrow is solvent-accessible in Dpo4. (BF PDB code: 1LV5, chains A/C/D [11]; Dpo4 PDB code: 2ATL, chains A/D/E [14]. )…”

“…Crystallographic structures of BF have been extensively studied and there is considerable understanding of the replication mechanism for unmodified DNA [11,12]. Likewise, X-ray crystallographic structures of Dpo4 in complex with DNA, both with and without dNTP have been determined (for example, see Refs [4,[13][14][15][16][17]).…”