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Mammalian DNA polymerase (pol ), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol copies undamaged DNA with average single-base substitution and deletion error rates of 7 ؋ 10 ؊3 and 2 ؋ 10 ؊3 , respectively. These error rates are high when compared to those of most other DNA polymerases. pol also has unusual error specificity, producing a high proportion of T⅐CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/ DinB family, pol can processively synthesize chains of 25 or more nucleotides. This moderate processivity may reflect a contribution of C-terminal residues, which include two zinc clusters. The very low fidelity and moderate processivity of pol is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.The recently discovered UmuC/DinB nucleotidyl transferase superfamily of DNA polymerases (1-3) includes a subfamily whose members share extensive amino acid sequence homology with the Escherichia coli dinB gene product. The dinB gene is required for untargeted mutagenesis of phage (4), and overexpression of dinB in E. coli increases the spontaneous mutation rate in plasmids, especially for single-base deletions in a run of guanine residues (5). The dinB gene encodes DNA polymerase (pol) IV, a distributive enzyme that lacks detectable 3Ј35Ј exonuclease activity (6). pol IV has limited ability to bypass UV radiation-induced photoproducts, but misinserts nucleotides at undamaged template sites at rates that are higher than those observed for DNA pol III, the major replicative enzyme in E. coli (7). Moreover, when incubated in the presence of a template-primer with a terminal mismatch with a potential for misalignment, pol IV generates DNA products that are one nucleotide shorter than expected (6). This is consistent with the Ϫ1 frameshift mutations seen when dinB is overexpressed (5).Orthologs of E. coli dinB have been identified in eukaryotes (2, 8). The human DINB1 gene is localized at chromosome 5q13 and encodes an 870-amino acid DNA polymerase (9 -11), which we refer to here as DNA polymerase (pol ).1 The product of the hDINB1 gene has also been designated DNA polymerase (10), a designation used earlier (12) for the human homolog of the Drosophila melanogaster mus308 gene. pol has several properties in common with E. coli pol IV. When purified from insect cells expressing the full-length polymerase fused to glutathione S-transferase (GST) (11) or purified as a catalytically active fragment of amino acids 1-560 (9), pol lacks detectable 3Ј35Ј exonuclease activity. The purified full-length GST fusion protein has optimal activity at 37°C over the pH range 6.5-7.5, it is insensitive to inhibition by aphidicolin or dideoxynucleotides, and Mg 2ϩ is preferred over Mn 2ϩ as the essential divalent cation (11). Neither the full-length GST-enzyme purified from yeast cells (10) nor truncated pol (9)...

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RNA polymerase transcription factor IIF (TFIIF) is required for initiation at most, if not all, polymerase II promoters. We report here the cloning and sequencing of genes for a yeast protein that is the homolog of mammalian TFIIF. This yeast protein, previously designated factor g, contains two subunits, Tfgl and Tfg2, both of which are required for transcription, essential for yeast cell viability, and whose sequences exhibit significant similarity to those of the mammalian factor. The yeast protein also contains a third subunit, Tfg3, which is less tightly associated and at most stimulatory to transcription, dispensable for cell viability, and has no known counterpart in mammalian TFIIF. Remarkably, the TFG3 gene encodes yeast TAF30, and furthermore, is identical to ANC1, a gene implicated in actin cytoskeletal function in vivo {Welch and Drubin 1994). Tfg3 is also a component of the recently described mediator complex (Kim et al. 1994), whose interaction with the carboxy-terminal repeat domain of RNA polymerase II enables transcriptional activation. Deletion of TFG3 results in diminished transcription in vivo.[Key Words: RNA polymerase II; TFIIF; TFIID; TAF; transcription; Saccharomyces cerevisiae] Received September 8, 1994~ revised version accepted October 11, 1994.Five purified proteins, termed factors a, b, d, e, and g, are required for promoter-dependent transcription by purified yeast RNA polymerase II . Factors a, b, and e are structurally and functionally related to human RNA polymerase II transcription factor (TF) TFIIE, TFIIH, and TFIIB, respectively (Gileadi et al. 1992;Tschochner et al. 1992;Feaver et al. 1994). Factor d can be replaced by recombinant TATA-binding protein (TBP;Flanagan et al. 1990) Weft, in prep.). Factor g was shown previously to resemble TFIIF in its content of two essential polypeptides and its avid binding to purified RNA polymerase II (Henry et al. 1992). There were, however, notable differences between the yeast and human factors, such as the larger size of the factor-g polypeptides (105 and 54 kD, as compared with 74 and 30 kD for human TFIIF; Sopta et al. 1985) and the presence of a third subunit (30 kD) in the yeast factor that appeared to stimulate transcription but not to be required for the process. We report here the cloning and sequencing of genes for factor-g polypeptides, which clarifies the relationship to TFIIF and opens the way to genetic analysis of the factor in yeast. Our findings further disclose a surprising connection beSCorresponding author. tween basal and regulatory factors, which may shed light on mechanisms of transcriptional activation. Results Genes for factor-g subunitsFactor g was purified to homogeneity, and the three subunits were separated by reverse phase chromatography as described previously (Henry et al. 1992). The individual subunits were treated with either trypsin or endoproteinase Lys-C, and amino acid sequences were derived from the resulting peptides. For the 30-kD subunit (p30), one peptide sequence of 34 residues (underlined in F...