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)...

To understand the mechanisms underlying mutagenesis in eukaryotes better, we have cloned mouse and human homologs of the Escherichia coli dinB gene. E. coli dinB encodes DNA polymerase IV and greatly increases spontaneous mutations when overexpressed. The mouse and human DinB1 amino acid sequences share significant identity with E. coli DinB, including distinct motifs implicated in catalysis, suggesting conservation of the polymerase function. These proteins are members of a large superfamily of DNA damage-bypass replication proteins, including the E. coli proteins UmuC and DinB and the Saccharomyces cerevisiae proteins Rev1 and Rad30. In a phylogenetic tree, the mouse and human DinB1 proteins specifically group with E. coli DinB, suggesting a mitochondrial origin for these genes. The human DINB1 gene is localized to chromosome 5q13 and is widely expressed.

actions responsible for the presumptive positioning of TF-IIIB upstream of vertebrate U6 RNA genes are unknown. The absence of A-and B-block promoter elements argues against the involvement of TFIIIC. The surprising discovery that in vitro transcription of vertebrate U6 genes requires the TATA-binding protein (TBP) subunit of RNAPII initiation factor TFIID (38, 51) and an RNAPII factor that stabilizes TBP binding to the TATA box, TFIIA (55), raises the possibility that TFIIIB is positioned by TBP and associated proteins bound to the vertebrate U6 gene TATA box.We have previously isolated the U6 RNA gene, SNR6, from the yeast Saccharomyces cerevisiae and found that unlike vertebrate U6 genes, it contains an essential B-block promoter element (7). The SNR6 B block is in a unique position, downstream of the coding region (see Fig. 1). A candidate A-block element in the conventional intragenic position was also identified, and it was suggested that the yeast U6 RNA gene binds TFIIIB by the same TFIIICdependent mechanism used by tRNA genes (7). However, the yeast U6 gene has upstream sequences similar to the vertebrate U6 gene TATA box and PSE (6)