Cells deficient in PARP-1 show an accelerated accumulation of DNA single strand breaks, but not AP sites, over the PARP-1-proficient cells exposed to MMS

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is a base excision repair (BER) protein that binds to DNA single strand breaks (SSBs) and subsequently synthesizes and transfers poly(ADP-ribose) polymers to various nuclear proteins. Numerous biochemical studies have implicated PARP-1 as a modulator of BER; however, the role of PARP-1 in BER in living cells remains unclear partly due to lack of accurate quantitation of BER intermediates existing in cells. Since DT40 cells, chicken B lymphocytes, naturally lack PARP-2, DT40 cells allow for the investigation of the PARP-1 null phenotype without confounding by PARP-2. To test the hypothesis that PARP-1 is necessary for efficient BER during methylmethane sulfonate (MMS) exposure in vertebrate cells, intact DT40 cells and their isogenic PARP-1 null counterparts were challenged with different exposure scenarios for phenotypic characterization. With chronic exposure, PARP-1 null cells exhibited sensitivity to MMS but with an acute exposure did not accumulate base lesions or AP sites to a greater extent than wild-type cells. However, an increase in SSB content in PARP-1 null cell DNA, as indicated by glyoxal gel electrophoresis under neutral conditions, suggested the presence of BER intermediates. These data suggest that during exposure, PARP-1 impacts the stage of BER after excision of the deoxyribosephosphate moiety from the 5' end of DNA strand breaks by polymerase beta.

Figure 1

Sensitivity of DT40 and PARP-1 null cells to MMS. Survival curves of DT40 (PARP-1 proficient), DT40-derived PARP-1 null cell, and PARP-1 null cells with ectopic expression of hPARP-1 exposed to MMS for 10 days. Each point represents the mean and S.D. (bars) from three independent experiments.

Figure 2

Measurement of roN7-meG as a marker of MMS exposure. Genomic DNA from DT40 and PARP-1 null cells exposed to 1 mM for up to 4 h was subjected to alkaline conditions to induce a ring-opened form of N7-meG for subsequent immuno-slot blot analysis. Each point represents the mean of four independent measurements. Bars indicate S.D.

Figure 3

Measurement of AP sites in DT40 and PARP-1 null cells exposed to MMS. Genomic DNA from DT40 and PARP-1 null cells exposed to 1 mM for up to 4 h was reacted with ARP for slot blot analysis of AP sites. Each point represents the mean of four independent measurements. Bars indicate S.D.

Figure 4

Depletion of intracellular NAD(P)H in DT40 and PARP-1 null DT40 cells. NAD(P)H levels in (A) DT40 and (B) PARP-1 null cells continuously exposed to various concentrations of MMS for up to 4 h. NAD(P)H depletion in (C) DT40 and PARP-1 null cells exposed to various MMS concentrations for 4 h in the presence or absence of 3-AB (10 mM).

Figure 5

Gel electrophoresis analysis of glyoxal denatured DNA from DT40 and PARP-1 null cells exposed to MMS. (A) Representative gel showing the migration of genomic DNA from wild-type DT40 (+) and PARP-1 null (−) cells exposed to 1 mM MMS for 1–4 hours. (B) Representative gel showing the migration of genomic DNA from wild-type DT40 (+) and PARP-1 null (−) cells exposed to various MMS concentrations for 4 h. (C) Comparison of tail moment values as determined by image analysis software between wild-type DT40 and PARP-1 null cells (**P<0.01, *P<0.05, n=3, t test).

Figure 5

Gel electrophoresis analysis of glyoxal denatured DNA from DT40 and PARP-1 null cells exposed to MMS. (A) Representative gel showing the migration of genomic DNA from wild-type DT40 (+) and PARP-1 null (−) cells exposed to 1 mM MMS for 1–4 hours. (B) Representative gel showing the migration of genomic DNA from wild-type DT40 (+) and PARP-1 null (−) cells exposed to various MMS concentrations for 4 h. (C) Comparison of tail moment values as determined by image analysis software between wild-type DT40 and PARP-1 null cells (**P<0.01, *P<0.05, n=3, t test).