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.

Human DNA polymerase eta (Pol eta) modulates susceptibility to skin cancer by promoting DNA synthesis past sunlight-induced cyclobutane pyrimidine dimers that escape nucleotide excision repair (NER). Here we have determined the efficiency and fidelity of dimer bypass. We show that Pol eta copies thymine dimers and the flanking bases with higher processivity than it copies undamaged DNA, and then switches to less processive synthesis. This ability of Pol eta to sense the dimer location as synthesis proceeds may facilitate polymerase switching before and after lesion bypass. Pol eta bypasses a dimer with low fidelity and with higher error rates at the 3' thymine than at the 5' thymine. A similar bias is seen with Sulfolobus solfataricus DNA polymerase 4, which forms a Watson-Crick base pair at the 3' thymine of a dimer but a Hoogsteen base pair at the 5' thymine (ref. 3). Ultraviolet-induced mutagenesis is also higher at the 3' base of dipyrimidine sequences. Thus, in normal people and particularly in individuals with NER-defective xeroderma pigmentosum who accumulate dimers, errors made by Pol eta during dimer bypass could contribute to mutagenesis and skin cancer.

One of the most common DNA lesions arising in cells is an apurinic/apyrimidinic (AP) site resulting from base loss. Although a template strand AP site impedes DNA synthesis, translesion synthesis (TLS) DNA polymerases can bypass an AP site. Because this bypass is expected to be highly mutagenic because of loss of base coding potential, here we quantify the efficiency and the specificity of AP site bypass by two Y family TLS enzymes, Sulfolobus solfataricus DNA polymerase 4 (Dpo4) and human DNA polymerase (Pol ). During a single cycle of processive DNA synthesis, Dpo4 and Pol bypass synthetic AP sites with 13-30 and 10 -13%, respectively, of the bypass efficiency for undamaged bases in the same sequence contexts. These efficiencies are higher than for the A family, exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I. We then determined AP site bypass specificity for complete bypass, requiring insertion or misalignment at the AP site followed by multiple incorporations using the aberrant primer templates. Although Dpo4, Pol , and Klenow polymerase have different fidelity when copying undamaged DNA, bypass of AP sites lacking A or G by all three polymerases is nearly 100% mutagenic. The majority (70 -80%) of bypass events made by all three polymerases are insertion of dAMP opposite the AP site. Single base deletion errors comprise 10 -25% of bypass events, with other base insertions observed at lower rates. Given that mammalian cells contain five polymerases implicated in TLS, and given that a large number of AP sites are generated per mammalian cell per day, even moderately efficient AP site bypass could be a source of substitution and frameshift mutagenesis in vivo.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.

A DNA lesion created by oxidative stress is 7,8-dihydro-8-oxo-guanine (8-oxoG). Because 8-oxoG can mispair with adenine during DNA synthesis, it is of interest to understand the efficiency and fidelity of 8-oxoG bypass by DNA polymerases. We quantify bypass parameters for two DNA polymerases implicated in 8-oxoG bypass, Pols δ and η. Yeast Pol δ and yeast Pol η both bypass 8-oxoG and misincorporate adenine during bypass. However, yeast Pol η is 10-fold more efficient than Pol δ, and following bypass Pol η switches to less processive synthesis, similar to that observed during bypass of a cis-syn thymine-thymine dimer. Moreover, yeast Pol η is at least 10-fold more accurate than yeast Pol δ during 8-oxoG bypass. These differences are maintained in the presence of the accessory proteins RFC, PCNA and RPA and are consistent with the established role of Pol η in suppressing ogg1-dependent mutagenesis in yeast. Surprisingly different results are obtained with human and mouse Pol η. Both mammalian enzymes bypass 8-oxoG efficiently, but they do so less processively, without a switch point and with much lower fidelity than yeast Pol η. The fact that yeast and mammalian Pol η have intrinsically different catalytic properties has potential biological implications.

When cyclobutane pyrimidine dimers stall DNA replication by DNA polymerase (Pol) delta or epsilon, a switch occurs to allow translesion synthesis by DNA polymerase eta, followed by another switch that allows normal replication to resume. In the present study, we investigate these switches using Saccharomyces cerevisiae Pol delta, Pol epsilon and Pol eta and a series of matched and mismatched primer templates that mimic each incorporation needed to completely bypass a cis-syn thymine-thymine (TT) dimer. We report a complementary pattern of substrate use indicating that enzymatic switching involving localized translesion synthesis by Pol eta and mismatch excision and polymerization by a major replicative polymerase can account for the efficient and accurate dimer bypass known to suppress sunlight-induced mutagenesis and skin cancer.

Xeroderma pigmentosum variant (XPV) patients with mutations in the DNA polymerase H (pol h) gene are hypersensitive to sunlight and have greatly increased susceptibility to sunlight-induced skin cancer. Consistent with the ability of Pol H to efficiently bypass UV light-induced cyclobutane pyrimidine dimers, XPV cells lacking Pol H 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 H functions, we generated Pol H-deficient mice. Mice homozygous for a null mutation in pol h 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 H-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 H indicate that Pol H-dependent bypass of cyclobutane pyrimidine dimers suppresses UV light-induced skin cancer in mice. Moreover, 37.5% of pol h 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 h may also have an increased risk of skin cancer. (Cancer Res 2006; 66(1): 87-94)

The apolipoprotein ε4 allele ( APOE4 ) is the strongest genetic risk factor for Alzheimer’s disease (AD). APOE4 carriers develop systemic metabolic dysfunction decades before showing AD symptoms. Accumulating evidence shows that the metabolic dysfunction accelerates AD development, including exacerbated amyloid-beta (Aβ) retention, neuroinflammation and cognitive decline. Therefore, preserving metabolic function early on may be critical to reducing the risk for AD. Here, we show that inulin increases beneficial microbiota and decreases harmful microbiota in the feces of young, asymptomatic APOE4 transgenic (E4FAD) mice and enhances metabolism in the cecum, periphery and brain, as demonstrated by increases in the levels of SCFAs, tryptophan-derived metabolites, bile acids, glycolytic metabolites and scyllo-inositol. We show that inulin also reduces inflammatory gene expression in the hippocampus. This knowledge can be utilized to design early precision nutrition intervention strategies that use a prebiotic diet to enhance systemic metabolism and may be useful for reducing AD risk in asymptomatic APOE4 carriers.