DNA polymerases need to be engineered to achieve optimal performance for biotechnological applications, which often require high fidelity replication when using modified nucleotides and when replicating difficult DNA sequences. These tasks are achieved for the bacteriophage T4 DNA polymerase by replacing leucine with methionine in the highly conserved Motif A sequence (L412M). The costs are minimal. Although base substitution errors increase moderately, accuracy is maintained for templates with mono- and dinucleotide repeats while replication efficiency is enhanced. The L412M substitution increases intrinsic processivity and addition of phage T4 clamp and single-stranded DNA binding proteins further enhance the ability of the phage T4 L412M-DNA polymerase to replicate all types of difficult DNA sequences. Increased pyrophosphorolysis is a drawback of increased processivity, but pyrophosphorolysis is curbed by adding an inorganic pyrophosphatase or divalent metal cations, Mn2+ or Ca2+. In the absence of pyrophosphorolysis inhibitors, the T4 L412M-DNA polymerase catalyzed sequence-dependent pyrophosphorolysis under DNA sequencing conditions. The sequence specificity of the pyrophosphorolysis reaction provides insights into how the T4 DNA polymerase switches between nucleotide incorporation, pyrophosphorolysis and proofreading pathways. The L-to-M substitution was also tested in the yeast DNA polymerases delta and alpha. Because the mutant DNA polymerases displayed similar characteristics, we propose that amino acid substitutions in Motif A have the potential to increase processivity and to enhance performance in biotechnological applications. An underlying theme in this chapter is the use of genetic methods to identify mutant DNA polymerases with potential for use in current and future biotechnological applications.

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The poly (ADP-ribose) polymerase, tankyrase 1, is an encouraging pharmacological target for cancer therapy. First discovered by its association with the TRF1, tankyrase 1 plays an important role in the maintenance of telomere length, sister telomere association, and mitotic spindle organization. More recently, interactions between tankyrase 1 and axin were shown to stabilize β-catenin and modulate Wnt signaling, enabling selective targeting of the Wnt pathway with tankyrase inhibitors. In addition, tankyrase 1 has also demonstrated potential as a target for synthetic lethality for BRCA-deficient cancers. Given the effect on these cancer-associated pathways, there is a growing interest for identifying tankyrase-specific inhibitors. Historical methods to evaluate tankyrase activity are not amenable for high throughput screening. Limited availability of the full length enzyme required radioactivity to attain the needed sensitivity. Truncation of tankyrase 1 improved solubility and increased availability, but the physiological response may not be equivalent to the full length enzyme. Many of these assays have also used modified substrates where molecules such as fluorescein or biotin are linked to NAD without characterizing the effects on enzyme activity. Most historical assays have also relied on measuring autoribosylation of tankyrase 1; whereas, transribosylation may be more relevant for assessing the activity of tankyrase 1 on other molecules. To address these issues, we have developed a novel, in vitro, high throughput assay for evaluating the transribosylation activity of tankyrase 1. This is accomplished using an ELISA format which semi-quantitatively detects poly (ADP-ribose) (PAR) deposited onto immobilized histone proteins by tankyrase 1. An anti-PAR monoclonal antibody, goat anti-mouse IgG-HRP conjugate, and HRP substrate generate a colorimetric or chemiluminescent signal, and the conversion of substrate correlates with tankyrase 1 activity. This assay ensures physiological significance by utilizing the full length enzyme and unmodified substrate in a transribosylation format, providing sensitivity down to pmols of enzyme. This assay is utilized to demonstrate the specificity of the tankyrase 1 inhibitor, XAV939, for tankyrase 1 compared to PARP1 and to reveal differential effects of activated DNA for full-length tankyrase 1 compared to the truncated enzyme. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3884. doi:1538-7445.AM2012-3884

The 96-Well CometChip System is a high-throughput platform to simultaneously treat and measure DNA damage induced by different treatments, or among different cell types on a single slide using the comet assay. The CometChip is a consumable consisting of specifically sized micron pores patterned into agarose layered on a treated microscope slide. Ninety-six (96) separate wells are created by inserting the CometChip into a reusable 96-Well CometChip System, a magnetically-sealed cassette suitable for tissue culture incubators. Cells added to each well are captured by gravity into micropores and excess cells aspirated leaving an array of non-overlapping cells. Multiple experimental conditions are performed in parallel by the addition of different chemicals to respective wells. Once treatment is complete the CometChip is removed from the cassette and processed using standard alkaline comet conditions and imaging systems. A distributable CometChip requires precision manufacturing to ensure robust and reproducible performance. CometChip tolerances were investigated and compared to normal comet using cryopreserved comet control cells with known levels of DNA damage. After setting specifications, 96-Well CometChips with 30 micron pores were distributed to evaluate both intra and inter-chip variations between laboratories. To assure experimental consistency between labs, magnetically sealable cassettes, cryopreserved control cells and identical electrophoresis systems were provided to each lab. Data is presented to demonstrate the feasibility of manufacturing the CometChip for reproducible results based on the coefficients of variance (CVs) obtained between different wells and laboratories. In addition, data will be presented demonstrating the absence of cross talk between CometChip wells using the 96-Well CometChip System. (Work was supported by R44ES021116). Citation Format: Sandra R. Woodgate, Clare Whittaker, Jay George, Robert W. Sobol, Sandy Schamus-Haynes, Bevin P. Engelward, Jing Ge. 96-Well CometChip validation for simultaneous treatment and measurement of DNA damage in a single platform. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2014-2379

The mechanism of action of many classes of chemotherapeutic agents involves either the repression of DNA repair or an increase in DNA damage. However the measurement of DNA damage levels within a cell has been notoriously difficult and current methods to asses DNA damage potential of new chemotherapeutics have major technical flaws. The single cell gel electrophoresis (SCGE) assay is a long-standing method for measuring levels of DNA damage within a cell. The principle of SCGE is that DNA damage can cause DNA strand breaks in cells. These breaks cause the relaxation of the compact highly supercoiled DNA. The application of an electric field while the cells are embedded in agarose allows damaged DNA to migrate faster than intact DNA. The more breaks in the DNA, the further it will travel in the agarose resulting in the formation of a “comet” tail, the size and length of which directly correlates to the amount of DNA damage. The SCGE method benefits from both technical simplicity and high sensitivity. The major drawback is the assay is extremely laborious, lacks appropriate controls and has poor reproducibility. We have recently overcome these drawbacks by developing a 96-well plate format of the SCGE, aptly named “CometChip”. The CometChip uses micro-pillar technology to create an agarose 96-well chip where each well has approximately 300 micro-wells used to capture individual cells. Using this technology we can incorporate multiple treatments, controls and time points on a single CometChip which can then be rapidly analyzed using a fluorescence based imaging instrument. The utility of the new technology was tested using 75 different chemical compounds considered either genotoxic, non-genotoxic or unknown. The compounds were tested on two lymphocyte cell lines with different p53 status to compare the accumulation and repair of DNA damage. We report that the CometChip gives highly reproducible and accurate results without loss of sensitivity. In the high throughput screening approach using multiple CometChip apparatus, we estimate the throughput of the assay to be approximately 10,000% greater than doing traditional slide based comet analysis. The massive increase in processivity brings new opportunity for large-scale compound screening. The increased sensitivity coupled with large sample sizes will allow researchers the option to measure minor changes in DNA damage with unparalleled accuracy. Citation Format: Peter Sykora, Sandra Woodgate, Jay George, Robert W. Sobol. Next generation high capacity DNA damage detection assay for chemotherapy and genotoxic compound screening. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3600.