DNA lesions can often block DNA replication, so cells possess specialized low-fidelity, and often error-prone, DNA polymerases that can bypass such lesions and promote replication of damaged DNA. The Saccharomyces cerevisiae RAD30 and human hRAD30A encode Pol eta, which bypasses a cis-syn thymine-thymine dimer efficiently and accurately. Here we show that a related human gene, hRAD30B, encodes the DNA polymerase Pol iota, which misincorporates deoxynucleotides at a high rate. To bypass damage, Pol iota specifically incorporates deoxynucleotides opposite highly distorting or non-instructional DNA lesions. This action is combined with that of DNA polymerase Pol zeta, which is essential for damage-induced mutagenesis, to complete the lesion bypass. Pol zeta is very inefficient in inserting deoxynucleotides opposite DNA lesions, but readily extends from such deoxynucleotides once they have been inserted. Thus, in a new model for mutagenic bypass of DNA lesions in eukaryotes, the two DNA polymerases act sequentially: Pol iota incorporates deoxynucleotides opposite DNA lesions, and Pol zeta functions as a mispair extender.

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 the hRAD30A gene, which encodes the DNA polymerase in humans, hPol. Of the eukaryotic DNA polymerases, only human Pol and its yeast counterpart have the ability to replicate DNA containing a cis-syn thymine-thymine (T-T) dimer. Here we measure the fidelity of hPol on all four nondamaged template bases and at each thymine residue of a cis-syn T-T dimer. Opposite all four nondamaged template bases, hPol misincorporates nucleotides with a frequency of ϳ10 ؊2-10 ؊3, and importantly, hPol synthesizes DNA opposite the T-T dimer with the same accuracy and efficiency as opposite the nondamaged DNA. The low fidelity of hPol 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 Saccharomyces cerevisiae RAD30 gene is involved in error-free bypass of UV-damaged DNA (1-3). RAD30 encodes a DNA polymerase, Pol, that can efficiently replicate DNA past a cis-syn thymine-thymine (T-T) 1 dimer in template DNA, and it does so by inserting two adenines across from the two thymines of the dimer. (4) Cells from the cancer-prone, variant form of xeroderma pigmentosum (XP-V) are much slower than normal cells in replicating DNA containing UV damage (5, 6), and XP-V cell-free extracts are deficient in replicating DNA past a cis-syn T-T dimer (7). The hRAD30A gene encodes the human counterpart of yeast RAD30, and mutations in hRAD30A cause XP-V (8, 9). Human Pol, like it's yeast counterpart, bypasses a cis-syn T-T dimer (9). Here, we determine the fidelity of hPol opposite all four nondamaged template bases, as well as opposite the T-T dimer, by measuring the steady state kinetics of deoxynucleotide incorporation. We find that hPol is a low fidelity DNA polymerase. Interestingly, hPol misincorporates deoxynucleotides opposite the T-T dimer with the same frequency as it does opposite the nondamaged template bases, ϳ10 Ϫ2 -10 Ϫ3 . These observations suggest that hPol has a flexible active site that is relatively insensitive to geometric distortions in DNA and that this property enables the enzyme to replicate past a T-T dimer. MATERIALS AND METHODSOverexpression of the hRAD30A Gene-To overexpress the human Pol protein (hPol), the hRAD30A gene was cloned in-frame with the glutathione S-transferase gene, which is under the control of a galactose-inducible phosphoglycerate promoter. The hRAD30A gene was amplified from the genomic clone GS21749 (8) by PCR using the primer oligonucleotides 5Ј-CCCTGAAGCTTGGATCCACATATGGCTACTGG-ACAGGATCGAGTGGTTGCTC-3Ј and 5Ј-CAGGGAATTCGGATCCAA-TATTAAATCCTACAGGCAAGCCTGAGGGCAGC-3Ј, which generate BamHI restriction sites 6 nucleotides upstream and 35 nucleotides downstream of the hRAD30A open reading frame, respectively. The wild type 1.3-kilobase pair SnaBI/NcoI DNA fragment from GS21749, which contains an internal portion of hRAD30A, was t...

Proliferating cell nuclear antigen (PCNA) plays critical roles in many aspects of DNA replication and replication-associated processes, including translesion synthesis, error-free damage bypass, break-induced replication, mismatch repair, and chromatin assembly. Since its discovery, our view of PCNA has evolved from a replication accessory factor to the hub protein in a large protein-protein interaction network that organizes and orchestrates many of the key events at the replication fork. We begin this review article with an overview of the structure and function of PCNA. We discuss the ways its many interacting partners bind and how these interactions are regulated by post-translational modifications such as ubiquitylation and sumoylation. We then explore the many roles of PCNA in normal DNA replication and in replication-coupled DNA damage tolerance and repair processes. We conclude by considering how PCNA can interact physically with so many binding partners to carry out its numerous roles. We propose that there is a large, dynamic network of linked PCNA molecules at and around the replication fork. This network would serve to increase the local concentration of all the proteins necessary for DNA replication and replication-associated processes and to regulate their various activities.

SUMMARY 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 non-classical polymerase. In eukaryotes, this polymerase exchange requires PCNA monoubiquitination. To better understand the polymerase exchange, we have developed a novel 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 via their canonical hydrophobic surface. Moreover, the attachment of ubiquitin does not change PCNA’s conformation. We propose that PCNA ubiquitination facilitates non-classical polymerase recruitment to the back of PCNA by forming a new binding surface for non-classical polymerases, consistent with a “tool belt” model of the polymerase exchange.

DNA polymerase eta (Poleta) is unique among eukaryotic DNA polymerases in its proficient ability to replicate through distorting DNA lesions, and Poleta synthesizes DNA with a low fidelity. Here, we use pre-steady-state kinetics to investigate the mechanism of nucleotide incorporation by Poleta and show that it utilizes an induced-fit mechanism to selectively incorporate the correct nucleotide. Poleta discriminates poorly between the correct and incorrect nucleotide at both the initial nucleotide binding step and at the subsequent induced-fit conformational change step, which precedes the chemical step of phosphodiester bond formation. This property enables Poleta to bypass lesions with distorted DNA geometries, and it bestows upon the enzyme a low fidelity.