Errol C. Friedberg

DNA damage and repair.

The aesthetic appeal of the DNA double helix initially hindered notions of DNA mutation and repair, which would necessarily interfere with its pristine state. But it has since been recognized that DNA is subject to continuous damage and the cell has an arsenal of ways of responding to such injury. Although mutations or deficiencies in repair can have catastrophic consequences, causing a range of human diseases, mutations are nonetheless fundamental to life and evolution.

The Y-Family of DNA Polymerases

We would like to thank Tomoo Ogi for generating the unrooted phylogenetic tree shown in Figure 1Figure 1 and Junetsu Ito for his comments on our proposal.

How nucleotide excision repair protects against cancer

Eukaryotic cells can repair many types of DNA damage. Among the known DNA repair processes in humans, one type — nucleotide excision repair (NER) — specifically protects against mutations caused indirectly by environmental carcinogens. Humans with a hereditary defect in NER suffer from xeroderma pigmentosum and have a marked predisposition to skin cancer caused by sunlight exposure. How does NER protect against skin cancer and possibly other types of environmentally induced cancer in humans?

Mouse Rev1 protein interacts with multiple DNA polymerases involved in translesion DNA synthesis

Polκ and Rev1 are members of the Y family of DNA polymerases involved in tolerance to DNA damage by replicative bypass [translesion DNA synthesis (TLS)]. We demonstrate that mouse Rev1 protein physically associates with Polκ. We show too that Rev1 interacts independently with Rev7 (a subunit of a TLS polymerase, Polζ) and with two other Y‐family polymerases, Polι and Polη. Mouse Polκ, Rev7, Polι and Polη each bind to the same ∼100 amino acid C‐terminal region of Rev1. Furthermore, Rev7 competes directly with Polκ for binding to the Rev1 C‐terminus. Notwith standing the physical interaction between Rev1 and Polκ, the DNA polymerase activity of each measured by primer extension in vitro is unaffected by the complex, either when extending normal primer‐termini, when bypassing a single thymine glycol lesion, or when extending certain mismatched primer termini. Our observations suggest that Rev1 plays a role(s) in mediating protein–protein interactions among DNA polymerases required for TLS. The precise function(s) of these interactions during TLS remains to be determined.

Suffering in silence: the tolerance of DNA damage

When cells that are actively replicating DNA encounter sites of base damage or strand breaks, replication might stall or arrest. In this situation, cells rely on DNA-damage-tolerance mechanisms to bypass the damage effectively. One of these mechanisms, known as translesion DNA synthesis, is supported by specialized DNA polymerases that are able to catalyse nucleotide incorporation opposite lesions that cannot be negotiated by high-fidelity replicative polymerases. A second category of tolerance mechanism involves alternative replication strategies that obviate the need to replicate directly across sites of template-strand damage.

Fidelity and processivity of DNA synthesis by DNA polymerase κ, the product of the human DINB1 gene

Mammalian DNA polymerase κ (pol κ), a member of the UmuC/DinB nucleotidyl transferase superfamily, has been implicated in spontaneous mutagenesis. Here we show that human pol κ 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 κ also has unusual error specificity, producing a high proportion of T·CMP mispairs and deleting and adding non-reiterated nucleotides at extraordinary rates. Unlike other members of the UmuC/DinB family, pol κ 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 κ is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.

The 19S Regulatory Complex of the Proteasome Functions Independently of Proteolysis in Nucleotide Excision Repair

The 26S proteasome degrades proteins targeted by the ubiquitin pathway, a function thought to explain its role in cellular processes. The proteasome interacts with the ubiquitin-like N terminus of Rad23, a nucleotide excision repair (NER) protein, in Saccharomyces cerevisiae. Deletion of the ubiquitin-like domain causes UV radiation sensitivity. Here, we show that the ubiquitin-like domain of Rad23 is required for optimal activity of an in vitro NER system. Inhibition of proteasomal ATPases diminishes NER activity in vitro and increases UV sensitivity in vivo. Surprisingly, blockage of protein degradation by the proteasome has no effect on the efficiency of NER. This establishes that the regulatory complex of the proteasome has a function independent of protein degradation.

Deletion of the Saccharomyces cerevisiae gene RAD30 encoding an Escherichia coli DinB homolog confers UV radiation sensitivity and altered mutability.

Abstract The dinB gene of Escherichia coli is an SOS-inducible gene of unknown function. Its mode of regulation and the amino acid sequence similarity of the predicted DinB protein to the UmuC protein of E. coli both suggest a role in cellular responses to DNA damage and probably in error-prone repair. Proteins with sequence similarity to DinB have been predicted from genes cloned from various prokaryotic and eukaryotic organisms, including Caenorhabditis elegans. Here we present the phenotypic characterization of a haploid Saccharomyces cerevisiae strain deleted for the ORF YDR419W, encoding a yeast DinB homolog. The deletion mutant is viable but is moderately sensitive to killing following exposure to ultraviolet (UV) radiation. Hence, we have named the gene RAD30. Steady-state levels of RAD30 transcripts are increased following UV irradiation. UV-induced locus-specific reversion of an ochre allele (arg4-17) is reduced in the rad30 deletion mutant. However, enhanced mutability was observed following treatment with the alkylating agent methylmethanesulfonate (MMS). Spontaneous mutability was also slightly increased. We conclude that RAD30 encodes an accessory function involved in DNA repair and mutagenesis. We speculate that the relatively weak phenotype and the opposite effects on mutability as a function of the type of DNA damage involved may derive from a functional redundancy of yeast proteins which facilitate replicative bypass of non-coding DNA lesions.

Novel DNA Polymerases Offer Clues to the Molecular Basis of Mutagenesis

An examination of available databases indicates the existence of an extended family of DinB-related proteins. These include E. coli UmuC, yeast Rev1, C. elegans F22B7.6, yeast Rad30, and the two human Rad30 homologs mentioned above. Additionally, mouse and human homologs of the E. coli dinB gene have been cloned (Gerlach et al. 1999xGerlach, V.L, Aravind, L, Gotway, G, Schultz, R.A, Koonin, E.V, and Friedberg, E.C. Proc. Natl. Acad. Sci. USA. 1999; in pressSee all ReferencesGerlach et al. 1999). All members of this superfamily share motifs with the known DNA polymerases Rad30 and DinB. These motifs contain invariant negatively charged residues that are likely involved in nucleotide polymerization, suggesting that DNA polymerase activity is common to the entire superfamily (Gerlach et al. 1999xGerlach, V.L, Aravind, L, Gotway, G, Schultz, R.A, Koonin, E.V, and Friedberg, E.C. Proc. Natl. Acad. Sci. USA. 1999; in pressSee all ReferencesGerlach et al. 1999).Clearly additional biochemical characterization lies ahead to identify similarities and differences among this class of enzymes and their role(s) in mutagenesis during replication of damaged and undamaged DNA. It will be particularly interesting to determine if different polymerases are selected to negotiate specific types of base damage in template DNA, and if so, how this selectivity is achieved to result in error-free or error-prone replication. Additionally, it will be important to address whether these polymerases operate in strict isolation, or participate in recruitment of the displaced replication machinery to the replication fork to allow continuation of normal DNA synthesis. Finally, do specialized polymerases such as pol η have absolute specificity for thymine–thymine dimers in template DNA? If so are cytosine–thymine, thymine–cytosine, and cytosine–cytosine dimers in DNA subject to error-free replicative bypass by other DNA polymerases, or must they be removed by DNA repair to avoid permanent replicative arrest? Based on the recent rapid strides in our understanding of the molecular basis of mutagenesis, one can optimistically look forward to the emergence of definitive answers to these questions in the near future.*To whom correspondence should be addressed (e-mail: friedberg.errol@pathology.swmed.edu).

Y-family DNA polymerases in mammalian cells

Eukaryotic genomes are replicated with high fidelity to assure the faithful transmission of genetic information from one generation to the next. The accuracy of replication relies heavily on the ability of replicative DNA polymerases to efficiently select correct nucleotides for the polymerization reaction and, using their intrinsic exonuclease activities, to excise mistakenly incorporated nucleotides. Cells also possess a variety of specialized DNA polymerases that, by a process called translesion DNA synthesis (TLS), help overcome replication blocks when unrepaired DNA lesions stall the replication machinery. This review considers the properties of the Y-family (a subset of specialized DNA polymerases) and their roles in modulating spontaneous and genotoxic-induced mutations in mammals. We also review recent insights into the molecular mechanisms that regulate PCNA monoubiquitination and DNA polymerase switching during TLS and discuss the potential of using Y-family DNA polymerases as novel targets for cancer prevention and therapy.