An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

An assay to measure poly(ADP ribose) glycohydrolase (PARG) activity in cells

After a DNA damage signal multiple polymers of ADP ribose attached to poly(ADP) ribose (PAR) polymerases (PARPs) are broken down by the enzyme poly(ADP) ribose glycohydrolase (PARG). Inhibition of PARG leads to a failure of DNA repair and small molecule inhibition of PARG has been a goal for many years. To determine whether biochemical inhibitors of PARG are active in cells we have designed an immunofluorescence assay to detect nuclear PAR after DNA damage. This 384-well assay is suitable for medium throughput high-content screening and can detect cell-permeable inhibitors of PARG from nM to µM potency. In addition, the assay has been shown to work in murine cells and in a variety of human cancer cells. Furthermore, the assay is suitable for detecting the DNA damage response induced by treatment with temozolomide and methylmethane sulfonate (MMS). Lastly, the assay has been shown to be robust over a period of several years.

The Smc5/6 Complex and Esc2 Influence Multiple Replication-associated Recombination Processes in Saccharomyces cerevisiae

Replication-associated recombinational repair is important for genome duplication and cell survival under DNA damage conditions. Several nonclassical recombination factors have been implicated in this process, but their functional relationships are not clear. Here, we show that three of these factors, Mph1, Mms2, and the Shu complex, can act independently to promote the formation of recombination intermediates during impaired replication. However, their functions become detrimental when cells lack the Smc5/6 complex or Esc2. We show that mph1Δ, mms2Δ, and shu1Δ suppress the sensitivity to the replication-blocking agent methylmethane sulfonate (MMS) in smc6 mutants, with double deletions conferring stronger suppression. These deletion mutations also rescue the MMS sensitivity of esc2Δ cells. In addition, two-dimensional gel analysis demonstrates that mph1Δ, mms2Δ, and shu1Δ each reduce the level of recombination intermediates in an smc6 mutant when cells replicate in the presence of MMS, and that double deletions lead to a greater reduction. Our work thus suggests that Mph1, Mms2, and the Shu complex can function in distinct pathways in replication-associated recombinational repair and that the Smc5/6 complex and Esc2 prevent the accumulation of toxic recombination intermediates generated in these processes.

Anti-clastogenic effect of b-glucan extracted from barley towards chemically induced DNA damage in rodent cells

b-Glucan (BG) was tested in vitro to determine its potential clastogenic and/or anti-clastogenic activity, and attempts were made to elucidate its possible mechanism of action by using combinations with an inhibitor of DNA polymerase. The study was carried out on cells deficient (CHO-k1) and cells proficient (HTC) in phases I and II enzymes, and the DNA damage was assessed by the chromosomal aberration assay. BG did not show a clastogenic effect, but was anti-clastogenic in both cell lines used, and at all concentrations tested (2.5, 5 and 10 mg/mL) in combination with damage inducing agents (methylmethane sulfonate in cell line CHO-k1, and methylmethane sulfonate or 2-aminoanthracene in cell line HTC). BG also showed a protective effect in the presence of a DNA polymerase b inhibitor (cytosine arabinoside-3-phosphate, Ara-C), demonstrating that BG does not act through an anti-mutagenic mechanism of action involving DNA polymerase b.

Identification and Characterization of a Second lexA Gene of Xanthomonas axonopodis Pathovar citri

ABSTRACT We previously identified and characterized a lexA gene from Xanthomonas axonopodis pv. citri. For this study, we cloned and expressed a lexA homologue from X. axonopodis pv. citri. This gene was designated lexA2, and the previously identified lexA gene was renamed lexA1. The coding region of lexA2 is 606 bp long and shares 59% nucleotide sequence identity with lexA1. Analyses of the deduced amino acid sequence revealed that LexA2 has structures that are characteristic of LexA proteins, including a helix-turn-helix DNA binding domain and conserved amino acid residues required for the autocleavage of LexA. The lexA2 mutant, which was constructed by gene replacement, was 4 orders of magnitude more resistant to the DNA-damaging agent mitomycin C at 0.1 μg/ml and 1 order of magnitude more resistant to another DNA-damaging agent, methylmethane sulfonate at 30 μg/ml, than the wild type. A lexA1 lexA2 double mutant had the same degree of susceptibility to mitomycin C as the lexA1 or lexA2 single mutant but was 1 order of magnitude more resistant to methylmethane sulfonate at 30 μg/ml than the lexA1 or lexA2 single mutant. These results suggest that LexA1 and LexA2 play different roles in regulating the production of methyltransferases that are required for repairing DNA damage caused by methylmethane sulfonate. A mitomycin C treatment also caused LexA2 to undergo autocleavage, as seen with LexA1. The results of electrophoresis mobility shift assays revealed that LexA2 does not bind the lexA1 promoter. It binds to both the lexA2 and recA promoters. However, neither LexA2 nor LexA1 appears to regulate recA expression, as lexA1, lexA2, and lexA1 lexA2 mutants did not become constitutive for recA transcription and RecA production. These results suggest that recA expression in X. axonopodis pv. citri is regulated by mechanisms that have yet to be identified.