The many faces of DNA polymerases: strategies for mutagenesis and for mutational avoidance.

T past year or so has witnessed the emergence of a plethora of prokaryotic and eukaryotic genes that are known or predicted to encode previously unidentified DNA polymerases (1–13). Some of these are members of an extended superfamily of prokaryotic and eukaryotic proteins called the UmuCyDinB nucleotidyl transferase superfamily, named after early discovered prokaryotic members (14, 15). This superfamily is represented presently by the UmuC, DinB, Rad30, and Rev1 subfamilies (14, 15). More recently identified polymerases resemble DNA replicative enzymes, and others seem to be related to the Pol b and terminal transferase proteins. A current working hypothesis is that when highly processive semiconservative DNA replication is arrested at lesions in DNA, the replicative machinery is displaced from the replication fork and replaced by these DNA polymerases. When the offending lesion has been bypassed successfully, the polymerase displacementyreplacement process is reversed, and the replication machinery continues high-fidelity, highly processive DNA synthesis. There is substantial evidence that some of these prokaryotic polymerases are involved in such replicative bypass (translesion synthesis or TLS) of damaged DNA. Hence, the mechanism or mechanisms by which these enzymes function predictably lie at the heart of some types of DNA damage-induced mutagenesis in organisms such as Escherichia coli. A primary example is UmuC protein, one of the members of the UmuC subfamily. This protein complexes with UmuD9 protein (a proteolytically processed form of UmuD) to form a UmuD92C complex that facilitates TLS in the presence of E. coli DNA polymerase III holoenzyme (1–5, 13). The UmuD92C complex alone (most likely the UmuC polypeptide) is now called DNA polymerase V of E. coli (16–18). More recent studies have shown that DNA polymerase V bypasses thymine–thymine dimers, [6-4] photoproducts, and abasic sites in a highly error-prone manner in vitro (19). Additionally, this enzyme has poor fidelity on undamaged DNA primertemplates, with error rates of 1023 to 1024 (19), substantially higher than those of most replicative DNA polymerases in vitro (13). In addition to DNA polymerase V, a polymerase called DNA polymerase IV of E. coli, the product of the dinB gene and a member of the DinB subfamily, was purified recently (20). DNA polymerase IV is devoid of 39 3 59 exonuclease activity and is strictly distributive in nature. Significantly, with respect to its role in spontaneous mutagenesis in vivo, the enzyme introduces frameshifts on misaligned primer-template substrates, resulting in 21 frameshift mutations (20). Most recently, it has been shown that a purified maltose-binding protein–DinB fusion protein is unable to bypass cis-syn thymine–thymine dimers, [6-4] photoproducts, or abasic sites in template DNA in vitro (19). Clearly, unlike DNA polymerase V, E. coli DNA polymerase IV is not able to support TLS at sites of chemically altered bases but may be able to support DNA synthesis at misaligned replication forks. The spotlight is now shifting to the functions of some of the eukaryotic genes and their polypeptide products, in particular the biochemical demonstration that they are indeed DNA polymerases, as well as a consideration of their fidelity, processivity, and ability to support various types of TLS. A consistent theme that is now beginning to emerge is that a number of these DNA polymerases have poor fidelity on normal DNA primer-templates. Interestingly, however, this property may be inconsequential most of the time in living cells, because these polymerases normally may not have access to undamaged DNA. Indeed, the property of limited fidelity may be essential for the primary (and possibly exclusive) biological function of some of these enzymes, which is to negotiate noninstructional types of template damage or conformational distortions to allow normal replication to continue. Furthermore, these enzymes can support this function with relative accuracy or inaccuracy and hence generate mutations or not at such noninstructional sites, depending on the type of lesion and the particular polymerase in question. However, it remains to be proven that the poor replicational fidelity on undamaged DNA of some of these DNA polymerases that are already partially characterized and possibly of others that remain to be characterized is not biologically consequential. Eukaryotic orthologs of the E. coli dinB gene have been identified in the genomes of the fission yeast Schizosaccharomyces pombe, Caenorhabditis elegans, mice, and humans (14, 15, 21). The budding yeast Saccharomyces cerevisiae lacks this gene, and there is no obvious dinB ortholog in the genome of Drosophila melanogaster, the sequence of which was completed recently. The human DINB1 and mouse Dinb1 cDNAs have been cloned and partially characterized (14, 21). In a recent issue of PNAS, Johnson et al. (22) have shown that the polypeptide product of the human DINB1 gene (which has been named pol k or pol u by different groups; see below) is a DNA polymerase with properties very similar to those of the orthologous enzyme from E. coli. Johnson et al. (22) purified a glutathione Stransferase (GST)–DinB1 fusion protein after overexpression in S. cerevisiae and demonstrated that polymerase activity was inactivated after mutation of highly conserved residues. Like the E. coli enzyme, GST–human DinB1 fusion protein can catalyze extension of a misaligned primer template to generate 21 frameshifts. Additionally, like its E. coli ortholog, human GST–DinB1 fusion protein is unable to replicate past cis-syn thymine– thymine dimers, [6-4] photoproducts, or abasic sites. Remarkably, the frequency of misincorporation at single nucleotide sites in undamaged primer-template DNA by