The DNA synthesome: its potential as a novel in vitro model system for studying S- phase specific anticancer agents  Waleed Abdel-Aziz, Linda H. Malkas, Philip W. Wills, Robert J. Hickey  Critical Reviews in Oncology / Hematology  Volume 48, Issue 1, Pages 19-33 (October 2003) DOI: 10.1016/S1040-8428(03)00096-9

Fig. 1 Current model of the mammalian cell DNA replication fork showing the minimal number of proteins required for DNA synthesis. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 2 Mammalian cell DNA synthesome. The model was described based on the observed fractionation, chromatographic, and sedimentation profiles of the individual replication proteins that copurify with the complex [13,14,22,33]. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 3 Neutral (A) and alkaline (B) gel analysis of the synthesome-mediated in vitro SV40 DNA replication products formed in the absence or presence of increasing concentrations of ara-CTP. Synthesome-driven in vitro DNA replication was performed according to Malkas et al. [22] and Abdel-Aziz et al. [29]. The DNA replication products were isolated by phenol/chloroform extraction followed by precipitation with 2-propanol in the presence of 2 M ammonium acetate. The isolated DNA was resuspended in 10 mM Tris/1 mM EDTA and electrophoresed through 1% agarose gels containing either TBE (A) (90 mM Tris/90 mM boric acid/2 mM EDTA) or alkaline (50 mM NaOH/1 mM EDTA) buffers. Gels were dried and exposed to Kodak XAR-5 films at −80°C for autoradiographic analysis of the resolved DNA replication products. ssl, single stranded linear DNA; ccc, covalently closed circular DNA. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 4 Neutral (A) and alkaline (B) gel analysis of the synthesome-mediated in vitro DNA replication products formed in the absence or presence of increasing concentrations of CPT. The DNA replication reactions were performed in the presence of increasing concentrations of CPT. Control experiments were carried out in the presence of DMSO. ssl, single-stranded linear DNA; ccc, covalently closed circular DNA. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 5 HPLC elution profile of nuclease-treated synthesome DNA replication products. Synthesome-mediated SV40 in vitro DNA replication was performed as described [22]. To determine the position of ara-CMP in newly synthesized DNA, the reactions used 3H-ara-CTP in place of dCTP. The DNA replication products were subjected to digestion by 50 U of micrococcal nuclease at 37°C for 45 min followed by two rounds of digestion each with 5 U of spleen phosphodiesterase at 37°C for 30 min and the products of digestion were resolved by HPLC analysis [40]. The elution positions of the 3H-ara-C nucleoside, the 3′ monophosphate nucleotide and the triphosphate nucleotide are indicated. These positions were assigned based on their coelution with authentic standards. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 6 Effects of BPU derivatives and ara-CTP on the synthesome-associated DNA polymerase α activity in Q-Sepharose fractions. The reaction was initiated by incubating the reaction mixture (20 μl) in the absence or presence of increasing concentrations of different BPU or ara-CTP (1, 10, 50, 100, 250, 500, and 1000 μM) for 1 h at 37°C. The reaction mixture was spotted onto DE81 filters and the amount of 3H-TTP retained on the filters was determined by liquid scintillation counting. Each point value represents the mean±S.E.M. of three to five experiments. Control reactions were performed in the presence of DMSO in place of BPU compounds. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 7 Synthesome-mediated in vitro DNA replication depends on the presence of the SV40 origin of replication. Synthesome-mediated in vitro DNA replication reactions were performed as described in the text using either pUC.HSO (ori+) or pUC.8-4 (ori−) plasmids. Lanes 1 and 2, synthesome-mediated in vitro DNA replication using pSVO+ plasmid in the absence or presence of T antigen, respectively; lanes 3, 4, 7, and 8, reactions performed using pUC.HSO (ori+) plasmid; lanes 5, 6, 9, and 10, reactions performed using pUC.8-4 (ori−) plasmid. MCF-7 and MDA MB-468 above the figure indicate the breast cancer cell line from which the DNA synthesome was isolated. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 8 (A) A map of the pSVO+ plasmid restriction sites with the replication origin (ori) indicated within the 900 bp fragment. (B) Agarose gel electrophoresis of the DNA digestion products after staining with ethidium bromide and illumination with UV light. Lane 1, a mixture of 100 bp and 1 kb DNA ladder; lane 2, the five DNA fragments produced after digestion of the plasmid with the restriction endonucleases; lane 3, pSVO+ plasmid (supercoiled) without digestion; lane 4, pSVO+ after its linearization by Ava I enzyme. The numbers beside the panel indicate the sizes of the DNA fragments in bp. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)

Fig. 9 Kinetic analyses of the synthesome-mediated in vitro DNA replication in the absence (A) or presence of ara-CTP (B) or CPT (C). The size of each individual DNA fragment formed after digestion with the restriction endonucleases indicated are given in base pairs (bp). In vitro SV40 origin-dependent DNA replication reactions were performed as described [15,22] in the absence or presence of ara-CTP or CPT. The reaction mixtures (500 μl) were incubated at 25°C for 2 h. Aliquots (50 μl) were removed at 0, 1, 3, 5, 10, 15, 30, 60, 90, and 120 min-intervals. DNA replication products were isolated by phenol/chloroform extraction and treated with Dpn I (2 U) for 1 h at 37°C. The Dpn I resistant products were purified with Chroma Spin Columns (Clontech Laboratories, CA) and treated with the combination of restriction endonucleases (PshA I, Ava I, AlwN I, Bsa I, and Sca I) for 1 h at 37°C followed by another hour at 50°C. The digestion products were resolved from one another by electrophoresis through a 5% polyacrylamide gel containing TBE buffer. Gels were dried and the positions of the reaction products were visualized by autoradiography using Kodak XAR-5 films at −80°C. Critical Reviews in Oncology / Hematology 2003 48, 19-33DOI: (10.1016/S1040-8428(03)00096-9)