Polymerase structures and function: variations on a theme?

Abstract

In recent years several lines of evidence have led to the proposal that all nucleic acid polymerases show fundamental similarities in structure and the mechanism of catalysis. First, protein sequence alignments suggested that all polymerases contain the same group of important side chains, which include two or, more commonly, three carboxylates. Second, crystallographic studies of four polymerases showed the polymerase domain of each enzyme folded to form a U-shaped cleft with the conserved carboxylates located in similar positions in the four structures. Finally, site-directed mutagenesis experiments have demonstrated that the conserved residues, particularly the carboxylates, play an important role in the polymerase reaction.The apparent similarity in the core polymerase structures cuts across the division of polymerases into classes based on whether the template and the synthesized strand are DNA or RNA. The implication is therefore that the basic mechanism of phosphoryl transfer is the same throughout the polymerase superfamily, although additional features must be present to account for the use of deoxyribo-or ribo-substrates in the four classes of polymerase. (This idea is consistent with the observation that polymerases can often be induced, by changing the reaction conditions, to use the ''wrong'' substrate.) A second obvious distinction between polymerase classes is the presence of additional functions: examples are the proofreading 3Ј-5Ј exonuclease of many DNA-dependent DNA polymerases, the 5Ј-3Ј exonuclease of some bacterial DNA polymerases, the RNase H activity of reverse transcriptases, and the binding of replicative polymerases to accessory proteins. However, there is reason to believe that these additional functions do not affect the basic properties of the polymerase core, since the known polymerase structures indicate a modular arrangement, with additional enzymatic activities present on independent structural domains.The four polymerase structures currently known represent four distinct polymerase families and three polymerase classes. The Klenow fragment of Escherichia coli DNA polymerase I (Pol I), a DNA-dependent DNA polymerase, is a monomer of 68 kDa which has, in addition to the polymerase domain, a separate structural domain containing the 3Ј-5Ј proofreading exonuclease (32). A second DNA-dependent DNA polymerase structure, that of rat DNA polymerase ␤ (Pol ␤), has recently been reported (14,40). This 39-kDa protein contains a separate 8-kDa N-terminal domain with single-stranded DNA binding and deoxyribose phosphate excision activities (27, 28, 30a) which has been proteolytically removed in some of the crystal forms. The reverse transcriptase from human immunodeficiency virus type 1 (HIV-1) is an example of an RNAdependent DNA polymerase (21,26). This enzyme is a heterodimer having one enzymatically active 66-kDa subunit containing polymerase and RNase H activities on separate domains. The second subunit (51 kDa), though derived from the same amino acid sequence as the polymerase domain...

Aminoglycoside binding to the hammerhead ribozyme: a general model for the interaction of cationic antibiotics with RNA 1 1Edited by J. Karn

“…Beside catalysts of the RNA world, enzymes catalyzing the polymerization of nucleic acids contain also two Mg 2 at a distance of 4 to 5 A Ê (Steitz & Steitz, 1993;Joyce & Steitz, 1995;Sousa, 1996). This was proven by X-ray crystallographical structure determination for DNA polymerase I from Escherichia coli (Beese & Steitz, 1991) and for the DNA polymerase b from rat (Pelletier et al, 1994).…”

3′-Azido-3′-deoxythymidine-resistant Mutants of DNA Polymerase β Identified by in Vivo Selection

“…The 31-kDa domain can be further subdivided into fingers, palm, and thumb domains by analogy to the shape of a hand. As in other polymerases, the active site is defined by two Mg 2ϩ ions which participate in the catalytic reaction; these atoms are held in place by three conserved acidic residues (5,18,19). In the case of pol ␤, the Mg We mapped the altered amino acids to the pol ␤ crystal structure (5, 6); both carry nonconservative substitutions located in a loop connecting two ␤ strands within the palm domain (Fig.…”

The Acidic Triad Conserved in Type IA DNA Topoisomerases Is Required for Binding of Mg(II) and Subsequent Conformational Change

“…Comparison of their polypeptide sequences showed that the conserved positions include the acidic residues Asp-111, 4). When the crystal structure of the 67-kDa Nterminal transesterification domain of the enzyme was published, it was noted (5) that these three acidic residues in the active site are arranged similarly to the three acidic residues known to coordinate two divalent ions in Klenow fragment (6) that are required for the nucleotidyl transfer activity (7,8). These residues are found in domain I of the 67-kDa structure (5), which is similar to the BЈ domain of the Saccharomyces cerevisiae DNA topoisomerase II structure (9).…”