Polymerase structures and function: variations on a theme?

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 deoxyriboor 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 39-59 exonuclease of many DNA-dependent DNA polymerases, the 59-39 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 39-59 proofreading exonuclease (32). A second DNA-dependent DNA polymerase structure, that of rat DNA polymerase b (Pol b), 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, is folded so as to preclude enzymatic activity (26); instead, this subunit may interact with the tRNA molecule that is used as a primer for reverse transcription. The fourth structure, that of the 99-kDa monomeric RNA polymerase from bacteriophage T7 (41), represents the class of DNA-dependent RNA polymerases. In addition to the polymerase domain, this molecule contains a separate N-terminal domain of about 300 amino acids that is probably involved in functions specific to RNA synthesis. For three of these polymerases (Klenow fragment, Pol b, and HIV-1 reverse transcriptase), cocrystal data provide information on the binding of DNA to the polymerase domain (4, 21, 34). In each case, the crystallographic data lead to a model entirely consistent with existing biochemical and molecular genetic data for that enzyme system. Nevertheless, others have suggested (1, 34) that some of the cocrystal structures are not relevant because of an apparent disagreement between the conclusions reached from these structures. We present here our contrasting view that this disagreement is the result of an inappropriate alignment of nonhomologous structures. When the structures are appropriately aligned, the expected similarities in substrate binding and catalysis at the polymerase site are preserved. In this article we shall ignore the other activities present in some polymerases in order to focus on the current understanding of how the polymerase domain itself is constructed and how catalysis of the polymerase reaction is achieved.