Xeroderma pigmentosum (XP) patients are highly sensitive to sunlight, and they suffer from a high incidence of skin cancers. The variant form of XP results from mutations in thehRAD30A gene, which encodes the DNA polymerase in humans, hPolη. Of the eukaryotic DNA polymerases, only human Polη and its yeast counterpart have the ability to replicate DNA containing acis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPolη on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPolη misincorporates nucleotides with a frequency of ∼10⁻²-10⁻³, and importantly, hPolη synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPolη may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.

The monoubiquitination of processivity factor PCNA at Lys164 in yeast leads to exchange of a replicative DNA polymerase with a translesion one. Now a functional, split version of monoubiquitinated PCNA is developed and its crystal structure shows that the ubiquitin moiety is located at the back face of the PCNA ring and does not cause large conformational changes in PCNA. DNA synthesis by classical polymerases can be blocked by many lesions. These blocks are overcome by translesion synthesis, whereby the stalled classical, replicative polymerase is replaced by a nonclassical polymerase. In eukaryotes this polymerase exchange requires proliferating cell nuclear antigen (PCNA) monoubiquitination. To better understand the polymerase exchange, we developed a means of producing monoubiquitinated PCNA, by splitting the protein into two self-assembling polypeptides. We determined the X-ray crystal structure of monoubiquitinated PCNA and found that the ubiquitin moieties are located on the back face of PCNA and interact with it through their canonical hydrophobic surface. Moreover, the attachment of ubiquitin does not change PCNA's conformation. We propose that PCNA ubiquitination facilitates nonclassical polymerase recruitment to the back of PCNA by forming a new binding surface for nonclassical polymerases, consistent with a 'tool belt' model of the polymerase exchange.

The yeast RAD30 gene functions in error-free replication of UV-damaged DNA, and RAD30 encodes a DNA polymerase, pol eta, that has the ability to efficiently and correctly replicate past a cis-syn-thymine-thymine dimer in template DNA. To better understand the role of pol eta in damage bypass, we examined its fidelity and processivity on nondamaged DNA templates. Steady-state kinetic analyses of deoxynucleotide incorporation indicate that pol eta has a low fidelity, misincorporating deoxynucleotides with a frequency of about 10(-2) to 10(-3). Also pol eta has a low processivity, incorporating only a few nucleotides before dissociating. We suggest that pol eta's low fidelity reflects a flexibility in its active site rendering it more tolerant of DNA damage, while its low processivity limits its activity to reduce errors.

Both in yeast and humans, DNA polymerase (Pol) η functions in the error-free replication of UV-damaged DNA, and Polη has the unique ability to efficiently replicate through a cis-syn thymine–thymine (T–T) dimer by inserting two As opposite the two Ts of the dimer. Although human DINB1 -encoded Polκ belongs to the same protein family as Polη, Polκ shows no ability to bypass this DNA lesion and its biological function has remained unclear. Here, we examine Polκ for its ability to extend from primer-terminal mispairs opposite nondamaged and damaged DNA templates. We find that Polκ is a promiscuous extender of primer-terminal mispairs opposite nondamaged DNA templates, and interestingly, it is also very efficient at extending from a G opposite the 3′T of a T–T dimer. These observations provide biochemical evidence for a role of Polκ in the extension of mismatched base pairs during normal DNA replication, and in addition, they implicate Polκ in the mutagenic bypass of T–T dimers. In its proficient mismatch extension ability, Polκ is more similar to the unrelated DNA polymerase ζ than it is to the phylogenetically related Polη or Polι. Thus, in humans, Polκ would compete with Polζ for the extension of mismatched base pairs on damaged and undamaged DNAs.