Lindsey Walsh

DNA-damage induction of RAD54 can be regulated independently of the RAD9- and DDC1-dependent checkpoints that regulate RNR2

Abstract. DNA damage checkpoints regulate a number of physiological responses after DNA damage. The transcriptional level of many genes is specifically induced in response to genotoxic stress in a checkpoint-dependent manner. The regulation of DNA damage-induced transcription of RAD54 and RNR2 by RAD9, DDC1, DUN1, CRT1 and MBP1 was investigated in Saccharomyces cerevisiae, using green fluorescent protein reporter assays and Northern blots. RAD54 and RNR2 reporter activity in response to the DNA damaging agent, methyl methanesulphonate, was measured in ddc1-Δ, rad9-Δ, ddc1-Δ/rad9-Δ, dun1-Δ, crt1-Δ and mbp1-Δ mutants and was compared with that of the wild type. RAD9 and DDC1 were shown to be required for a full RNR2 transcriptional response, although with the double mutant, ddc1-Δ/rad9-Δ, no additive effect on RNR2 induction was observed. RAD54 promoter activity was not significantly reduced in either rad9-Δ or ddc1-Δ mutants and was only partially reduced in the rad9-Δ/ddc1-Δ strain, suggesting that DNA damage induction of RAD54 must depend on other genes, in addition to RAD9 and DDC1. In the dun1-Δ mutant, RNR2 promoter activity was lowered, whilst that of RAD54 was increased, confirming that DUN1 is required for transcriptional induction of RNR2, but is not required for damage-induced transcription of RAD54. Analysis of the crt1-Δ strain confirmed that RNR2 is regulated via the CRT1 repressor pathway, downstream of DUN1, but RAD54 is not. MBP1 was shown to be required for transcription of RNR2, but was not needed for transcription of RAD54. These results indicate that RNR2 and RAD54 are regulated in different ways.

Target-assembled ExciProbes: Application to DNA Detection at the Level of PCR Product and Plasmid DNA

Abstract Recently, we introduced a novel exciplex-based approach for detection of nucleic acids using a model DNA-mounted exciplex system, consisting of two 8-mer ExciProbes hybridized to a complementary 16-mer DNA target. We now show, for the first time, that this approach can be used to detect DNA at the level of PCR product and plasmid, when the target sequence (5′-GCCAAACACAGAATCG-3′) was embedded in long DNA molecules (PCR products and ∼3 Kbp plasmid). A remarkably stringent demand is made of the solvent conditions for this exciplex emission to occur, viz., emission is optimal for DNA at 80% tri-fluoroethanol, even in the plasmid situations, raising the question of the molecular structural basis of this system. We show that a perfectly matched plasmid target can be differentiated from target containing single nucleotide substitutions; hence, ExciProbes could be applied to SNP analysis. The effect of counter cations (Na+, K+, and Mg2+) and PCR additives on exciplex emission has been also examined.

SNP detection for cytochrome P450 alleles by target-assembled tandem oligonucleotide systems based on exciplexes

Abstract We report the first use of exciplex-based split-probes for detection of the wild type and *3 mutant alleles of human cytochrome P450 2C9. A tandem 8-mer split DNA oligonucleotide probe system was designed that allows detection of the complementary target DNA sequence. This exciplex-based fluorescence detector system operates by means of a contiguous hybridization of two oligonucleotide exciplex split-probes to a complementary target nucleic acid target. Each probe oligonucleotide is chemically modified at one of its termini by a potential exciplex-forming partner, each of which is fluorescently silent at the wavelength of detection. Under conditions that ensure correct three-dimensional assembly, the chemical moieties on suitable photoexcitation form an exciplex that fluoresces with a large Stokes shift (in this case 130 nm). Preliminary proof-of-concept studies used two 8-mer probe oligonucleotides, but in order to give better specificity for genomic applications, probe length was extended to give coverage of 24 bases. Eight pairs of tandem 12-mer oligonucleotide probes spanning the 2C9*3 region were designed and tested to find the best set of probes. Target sequences tested were in the form of (i) synthetic oligonucleotides, (ii) embedded in short PCR products (150 bp), or (iii) inserted into plasmid DNA (∼ 3 Kbp). The exciplex system was able to differentiate wild type and human cytochrome P450 2C9 *3 SNP (1075 A→C) alleles, based on fluorescence emission spectra and DNA melting curves, indicating promise for future applications in genetic testing and molecular diagnostics.

New concepts of fluorescent probes for specific detection of DNA sequences: bis-modified oligonucleotides in excimer and exciplex detection.

The detection of single base mismatches in DNA is important for diagnostics, treatment of genetic diseases, and identification of single nucleotide polymorphisms. Highly sensitive, specific assays are needed to investigate genetic samples from patients. The use of a simple fluorescent nucleoside analogue in detection of DNA sequence and point mutations by hybridisation in solution is described in this study. The 5′-bispyrene and 3′-naphthalene oligonucleotide probes form an exciplex on hybridisation to target in water and the 5′-bispyrene oligonucleotide alone is an adequate probe to determine concentration of target present. It was also indicated that this system has a potential to identify mismatches and insertions. The aim of this work was to investigate experimental structures and conditions that permit strong exciplex emission for nucleic acid detectors, and show how such exciplexes can register the presence of mismatches as required in SNP analysis. This study revealed that the hybridisation of 5′-bispyrenyl fluorophore to a DNA target results in formation of a fluorescent probe with high signal intensity change and specificity for detecting a complementary target in a homogeneous system. Detection of SNP mutations using this split-probe system is a highly specific, simple, and accessible method to meet the rigorous requirements of pharmacogenomic studies. Thus, it is possible for the system to act as SNP detectors and it shows promise for future applications in genetic testing.