Evidence for Abasic Site Sugar Phosphate-Mediated Cytotoxicity in Alkylating Agent Treated <em>Saccharomyces cerevisiae</em>

<div><p>To better understand alkylating agent-induced cytotoxicity and the base lesion DNA repair process in <em>Saccharomyces cerevisiae</em>, we replaced the <em>RAD27^FEN1</em> open reading frame (ORF) with the ORF of the bifunctional human repair enzyme DNA polymerase (Pol) β. The aim was to probe the effect of removal of the incised abasic site 5′-sugar phosphate group (i.e., 5′-deoxyribose phosphate or 5′-dRP) in protection against methyl methanesulfonate (MMS)-induced cytotoxicity. In <em>S. cerevisiae</em>, Rad27^Fen1 was suggested to protect against MMS-induced cytotoxicity by excising multinucleotide flaps generated during repair. However, we proposed that the repair intermediate with a blocked 5′-end, i.e., 5′-dRP group, is the actual cytotoxic lesion. In providing a 5′-dRP group removal function mediated by dRP lyase activity of Pol β, the effects of the 5′-dRP group were separated from those of the multinucleotide flap itself. Human Pol β was expressed in <em>S. cerevisiae</em>, and this partially rescued the MMS hypersensitivity observed with <em>rad27^fen1</em>-null cells. To explore this rescue effect, altered forms of Pol β with site-directed eliminations of either the 5′-dRP lyase or polymerase activity were expressed in <em>rad27^fen1</em>-null cells. The 5′-dRP lyase, but not the polymerase activity, conferred the resistance to MMS. These results suggest that after MMS exposure, the 5′-dRP group in the repair intermediate is cytotoxic and that Rad27^Fen1 protection against MMS in wild-type cells is due to elimination of the 5′-dRP group.</p> </div

Mammalian Base Excision Repair: Functional Partnership between PARP-1 and APE1 in AP-Site Repair

<div><p>The apurinic/apyrimidinic- (AP-) site in genomic DNA arises through spontaneous base loss and base removal by DNA glycosylases and is considered an abundant DNA lesion in mammalian cells. The base excision repair (BER) pathway repairs the AP-site lesion by excising and replacing the site with a normal nucleotide via template directed gap-filling DNA synthesis. The BER pathway is mediated by a specialized group of proteins, some of which can be found in multiprotein complexes in cultured mouse fibroblasts. Using a DNA polymerase (pol) β immunoaffinity-capture technique to isolate such a complex, we identified five tightly associated and abundant BER factors in the complex: PARP-1, XRCC1, DNA ligase III, PNKP, and Tdp1. AP endonuclease 1 (APE1), however, was not present. Nevertheless, the complex was capable of BER activity, since repair was initiated by PARP-1’s AP lyase strand incision activity. Addition of purified APE1 increased the BER activity of the pol β complex. Surprisingly, the pol β complex stimulated the strand incision activity of APE1. Our results suggested that PARP-1 was responsible for this effect, whereas other proteins in the complex had no effect on APE1 strand incision activity. Studies of purified PARP-1 and APE1 revealed that PARP-1 was able to stimulate APE1 strand incision activity. These results illustrate roles of PARP-1 in BER including a functional partnership with APE1.</p></div

Effect of purified BER factors on APE1-independent BER by the pol β complex.

<p>(A) A schematic representation of the DNA substrate containing the AP-site and the reaction scheme is shown. (B) BER activity of the pol β complex was evaluated on an AP- site-containing DNA substrate by measuring incorporation of [α-³²P]dCMP as a function of different components in the reaction mixture and incubation time. Reaction conditions and product analysis are described under Materials and Methods. AP-site DNA was incubated with the pol β complex in the presence (+) or absence (-) of purified BER factors including PARP-1 XRCC1, PNKP, DNA ligase I, as indicated at the top of the phosphorimage. Lane 13 represents the result after incubation of the reaction mixture without the pol β complex or purified proteins. Incubation was at 37°C for 15 and/or 30 min. The reaction products were separated by electrophoresis in a 16% polyacrylamide gel containing 8 M urea. A Typhoon PhosphorImager was used for gel scanning and imaging. The positions of the unligated BER product and ligated BER product are indicated. (C) AP-site DNA was incubated with the pol β complex in the presence (+) or absence (-) of purified BER factors, as indicated below the histogram. The ligated and unligated BER products at 30 min incubation were quantified using ImageQuant software and plotted in a histogram. The grey and black bars represent unligated and ligated BER products, respectively. (D) A histogram illustrating the ratios of ligated BER product to total BER products (both ligated plus unligated BER products) is shown.</p

<i>In vitro</i> primer extension assay by <i>S. cerevisiae</i> extracts.

<p>(A) Schematic representations of the substrate and the reaction scheme are shown. (B) The primer extension reaction mixture was incubated either with reaction mixture alone (lane 1), or with extracts from <i>wild-type</i> (lanes 2–4), <i>rad27</i>Δ (lanes 5–7), <i>rad27::POLβ</i> (lanes 8–10), <i>rad27::polβ-D256A</i> (lanes 11–13), <i>rad27::polβ-3K</i> (lanes14–16), and purified pol β (lanes 17–19), respectively. Reaction mixtures were incubated at 35°C and samples were withdrawn at the indicated periods. After incubation, the reaction products were processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047945#pone-0047945-g003" target="_blank">Figure 3</a>. The positions of ³²P-primer and the extension products are indicated.</p

Effect of PARP-1 on the steady-state rate of AP-site incision catalyzed by APE1.

<p>The DNA substrate with THF (100 nM) was preincubated with 25–500 nM PARP-1. After adding 0.5 nM APE1, the reaction mixture was incubated for 10 s to 5 min at 37°C. The reaction conditions and data analysis are described in Materials and Methods. The data representing the reaction products were fitted to an exponential equation to determine the steady-state rate of the APE1 incision reaction in the absence and presence of PARP-1. The average from three repeats is represented.</p

A model illustrating APE1-dependent and-independent mammalian BER coordinated by BER factors in the pol β complex.

<p>AP-site lesions in DNA that are formed by spontaneous hydrolysis of the <i>N</i>-glycosylic bond or by removal of inappropriate bases by DNA <i>N</i>-glycosylases are recognized by PARP-1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.ref044" target="_blank">44</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.ref050" target="_blank">50</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.ref061" target="_blank">61</a>]. By virtue of the presence of PARP-1 in the pol β complex, the complex is recruited to the AP-site in DNA. Upon binding to AP-site, PARP-1 is auto-poly(ADP-ribosyl)ated [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.ref044" target="_blank">44</a>]. While the complex remains bound to the AP-site DNA strand, BER may proceed either by an APE1-dependent (<i>left-hand</i> side of the scheme) or APE1-independent (<i>right-hand</i> side of the scheme) pathway. In the case of the APE1-dependent pathway, APE1 incises the AP-site, while the complex is still bound to the AP-site. The dRP removal, DNA synthesis and ligation steps are conducted. On the other hand, in situations where APE1 is deficient, APE1-independent BER operates where PNKP plays a central role [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.ref060" target="_blank">60</a>]. In this case, for example, the complex bound at the AP-site incises the DNA strand by its PARP-1’s lyase activity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.ref050" target="_blank">50</a>]. Tdp1 and/or PNKP trim or edit the 3′blocked group to generate the 3′-OH necessary for the DNA synthesis and ligation steps, respectively. PAPR-1 is depicted as the blue triangle in the pol β complex.</p

Hiding in Plain Sight: The Bimetallic Magnesium Covalent Bond in Enzyme Active Sites

The transfer of phosphate groups is an essential function of many intracellular biological enzymes. The transfer is in many cases facilitated by a protein scaffold involving two closely spaced magnesium “ions”. It has long been a mystery how these “ions” can retain their closely spaced positions throughout enzymatic phosphate transfer: Coulomb’s law would dictate large repulsive forces between these ions at the observed distances. Here we show, however, that the electron density can be borrowed from nearby electron-rich oxygens to populate a bonding molecular orbital that is largely localized between the magnesium “ions”. The result is that the Mg–Mg core of these phosphate transfer enzymes is surprisingly similar to a metastable [Mg₂]²⁺ ion in the gas phase, an ion that has been identified experimentally and studied with high-level quantum-mechanical calculations. This similarity is confirmed by comparative computations of the electron densities of [Mg₂]²⁺ in the gas phase and the Mg–Mg core in the structures derived from QM/MM studies of high-resolution X-ray crystal structures. That there is a level of covalent bonding between the two Mg “ions” at the core of these enzymes is a novel concept that enables an improved vision of how these enzymes function at the molecular level. The concept is broader than magnesiumother biologically relevant metals (e.g., Mn and Zn) can also form similar stabilizing covalent Me–Me bonds in both organometallic and inorganic crystals

Effect of PARP-1 on APE1-dependent BER.

<p>(A) A schematic representation of the DNA substrate containing the AP-site and the reaction scheme is shown. The BER reaction conditions and product analysis are described under Materials and Methods. (B) The BER reaction mixtures containing purified proteins XRCC1, PNKP, DNA ligase I and APE1 were supplemented either with PARP-1 (lanes 1–3) or dilution buffer (lanes 4–6). Repair was initiated by transferring the reaction mixtures to 37°C. Aliquots were withdrawn at 5, 10 and 20 min. The reaction products were analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124269#pone.0124269.g001" target="_blank">Fig 1</a>. The positions of the BER intermediate (unligated) and ligated BER products are indicated. (C) Quantification of the BER products was performed using ImageQuant software and data plotted as a function of incubation time (min). The plot demonstrates that BER product formation was linear during the 20 min incubation and that PARP-1 stimulated BER at least 2-fold as compared to the reaction without additional PARP-1.</p

Human <i>POLβ</i> rescues MMS sensitivity in <i>S. cerevisiae</i>, null for <i>rad27</i>.

<p>Experiments were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047945#s2" target="_blank">Materials and Methods</a>. Briefly, ten-fold dilutions (as shown) were set-up for each strain and culture (5 µl of each dilution) were spotted onto freshly prepared YPDA plates containing the indicated concentration of MMS. Photographs were taken on day 3 or 4 after plating. (A) Mock treatment, lanes 1–4; 0.5 mM MMS treatment, lanes 5–8; 1 mM MMS treatment, lanes 9–12. (B) Rescue of MMS sensitivity by human Pol β dRP lyase. Sensitivity to MMS was scored by spotting cultures of various strains on plates either without (mock) or with 1 mM of MMS. Resistance to MMS was observed in <i>rad27</i> null strains that contain <i>POLβ</i> or <i>polβ-D256A</i>. Lack of cell growth was observed only in the <i>rad27</i> null strains (<i>rad27</i>Δ) or the strain containing mutations in the dRP lyase domain (<i>rad27</i>::<i>polβ-3K</i>). Strains are indicated on the left.</p

<i>In vitro</i> BER capacity of <i>S. cerevisiae</i> extracts.

<p>(A) Repair reactions were incubated either with extracts from <i>wild-type</i> (lanes 1–4), <i>rad27</i>Δ (lanes 5–8), <i>rad27::POLβ</i> (lanes 9–10), <i>rad27::polβ-D256A</i> (lanes 11–14), or <i>rad27::polβ-3K</i> (lanes15–16), respectively. Note that all the reaction mixtures were supplemented with human DNA ligase I (200 nM) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047945#pone-0047945-g004" target="_blank">Figure 4</a>. Reaction mixtures were incubated at 35°C and samples were withdrawn at the indicated periods. After incubation, the reaction products were processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047945#pone-0047945-g003" target="_blank">Figure 3</a>. The positions of ligated BER product and un-ligated BER intermediate are indicated. (B) Lanes 1 to 8 from the panel (A) were exposed for a longer time to observe BER products in the <i>wild-type</i> (lanes 1–4) and <i>rad27</i>Δ (lanes 5–8) strains.</p