Scott D. McCulloch

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

In their seminal publication describing the structure of the DNA double helix 1, Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.

Increased Susceptibility to UV-Induced Skin Carcinogenesis in Polymerase {eta}-deficient Mice

Xeroderma pigmentosum variant (XPV) patients with mutations in the DNA polymerase eta (pol eta) gene are hypersensitive to sunlight and have greatly increased susceptibility to sunlight-induced skin cancer. Consistent with the ability of Pol eta to efficiently bypass UV light-induced cyclobutane pyrimidine dimers, XPV cells lacking Pol eta have diminished capacity to replicate UV-damaged DNA and are sensitive to UV light-induced killing and mutagenesis. To better understand these and other Pol eta functions, we generated Pol eta-deficient mice. Mice homozygous for a null mutation in pol eta are viable, fertile, and do not show any obvious spontaneous defects during the first year of life. However, fibroblasts derived from these mutant mice are sensitive to killing by exposure to UV light, and all Pol eta-deficient mice develop skin tumors after UV irradiation, in contrast to the wild-type littermate controls that did not develop such tumors. These results and biochemical studies of translesion synthesis by mouse Pol eta indicate that Pol eta-dependent bypass of cyclobutane pyrimidine dimers suppresses UV light-induced skin cancer in mice. Moreover, 37.5% of pol eta heterozygous mice also developed skin cancer during 5 months after a 5-month exposure to UV light, suggesting that humans who are heterozygous for mutations in pol eta may also have an increased risk of skin cancer.

Bi-directional Processing of DNA Loops by Mismatch Repair-dependent and -independent Pathways in Human Cells

Previous work has shown that small DNA loop heterologies are repaired not only through the mismatch repair (MMR) pathway but also via an MMR-independent pathway in human cells. However, how DNA loop repair is partitioned between these pathways and how the MMR-independent repair is processed are not clear. Using a novel construct that completely and specifically inhibits MMR in HeLa extracts, we demonstrate here that although MMR is capable of bi-directionally processing DNA loops of 2, 4, 5, 8, 10, or 12 nucleotides in length, the repair activity decreases with the increase of the loop size. Evidence is presented that the largest loop that the MMR system can process is 16 nucleotides. We also show that strand-specific MMR-independent loop repair occurs for all looped substrates tested and rigorously demonstrate that this repair is bi-directional. Analysis of repair intermediates generated by the MMR-independent pathway revealed that although the processing of looped substrates with a strand break 5′ to the heterology occurred similarly to MMR (i.e. excision is conducted by exonucleases from the pre-existing strand break to the heterology), the processing of the heterology in substrates with a 3′ strand break is consistent with the involvement of endonucleases.

Efficiency, Fidelity and Enzymatic Switching DurinG Translesion DNA Synthesis

More than half of the 16 human DNA polymerases may have some role in DNA replication and potentially modulate the biological effects of DNA template lesions that impede replication fork progression. As one approach to understand how multiple polymerases are coordinated at the fork, we recently quantified the efficiency and fidelity with which one particular translesion synthesis enzyme, human DNA polymerase ?, copies templates containing cis-syn thymine dimers. Several observations from that study were unanticipated. Here we discuss the structural and biological implications of those results in light of earlier studies of translesion synthesis.

Nick-dependent and independent processing of large DNA loops in human cells

DNA loop heterologies are products of normal DNA metabolism and can lead to severe genomic instability if unrepaired. To understand how human cells process DNA loop structures, a set of circular heteroduplexes containing a 30-nucleotide loop were constructed and tested for repair in vitro by human cell nuclear extracts. We demonstrate here that, in addition to the previously identified 5′ nick-directed loop repair pathway (Littman, S. J., Fang, W. H., and Modrich, P. (1999) J. Biol. Chem. 274, 7474–7481), human cells can process large DNA loop heterologies in a loop-directed manner. The loop-directed repair specifically removes the loop structure and occurs only in the looped strand, and appears to require limited DNA synthesis. Like the nick-directed loop repair, the loop-directed repair is independent of many known DNA repair pathways, including DNA mismatch repair and nucleotide excision repair. In addition, our data also suggest that an aphidicolin-sensitive DNA polymerase is involved in the excision step of the nick-directed loop repair pathway.

Measuring the fidelity of translesion DNA synthesis.

A method is described to measure the fidelity of copying past a DNA lesion in a defined sequence on a synthetic oligonucleotide primer-template. The DNA product is the result of a complete lesion bypass reaction, i.e., containing all four deoxynucleotide triphosphates and requiring both insertion opposite the lesion and multiple extensions from the resulting primer termini containing the lesion. The nascent strand is recovered and hybridized to a gapped region of the lacZalpha complementation gene of the M13mp2 genome. When this DNA is introduced into Escherichia coli, errors made during translesion DNA synthesis are detected by M13 plaque colors. Sequencing of DNA from mutant plaques defines the types of errors and permits calculation of error rates for base substitutions, insertions, and deletions. The method is illustrated here for bypass of a cis-syn thymine-thymine dimer by human DNA polymerase eta. The assay can be used with other lesions in various sequence contexts and with other polymerases with or without accessory proteins.

DNA Mismatch Repair in Tumor Suppression

It is believed that cancer is caused by mutations (1). In addition to the mutations induced by DNA damage, mutations can arise from mismatched base pairs (bps) generated during DNA replication and recombination. However, to avoid mutagenesis, cells possess mutation avoidance systems, one of which is the DNA mismatch repair (MMR) pathway. MMR is the primary cellular pathway that is responsible for correcting mispairs that arise during normal DNA metabolism. In bacteria, the importance of MMR in maintaining genomic stability was demonstrated with the observation, made more than 25 years ago, that defects in this pathway lead to elevated spontaneous mutability (2, 3). Inactivation of MMR in eukaryotic cells results in a mutator phenotype, and thus MMR is also crucial in maintaining genomic stability in eukaryotes. A dramatic example of this fact is the direct association of MMR deficiency with human hereditary and sporadic cancer.