Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA

Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin.     

Bleomycin-induced alterations in DNA replication: relationship to DNA damage

Bleomycin (BLM), a well-known DNA scission agent, is assumed to inhibit intracellular DNA replication by damaging the DNA template (cis-acting mechanism), although other DNA damaging compounds can alter DNA replication through modulation of crucial replication factor(s) (trans-acting mechanism). The present study examines the relationship between DNA damage and inhibition of replication caused by BLM in the well-defined simian virus 40 (SV40) intracellular and cell-free in vitro systems. Treatment of SV40-infected BSC-1 cells for 2 h with BLM at 50 microg/mL, induced 0.3 break/viral genome. Under the same treatment conditions, analysis of replication intermediates on two-dimensional gels showed a decrease in both mass of SV40 replication intermediates and replication activity. The mass of SV40 intermediates was decreased to about 30%, whereas replication activity was reduced to less than 5%. These results suggest that BLM inhibits both initiation and elongation phases of SV40 replication. In a cell-free DNA replication system, extracts from BLM-treated cells (50 micro/mL) were able to support SV40 DNA replication by only 50%. In this study, non-drug-treated DNA template was used, implying that BLM can induce a trans-acting effect. Finally, the drug-induced effects on SV40 DNA replication in cell-free and intracellular viral systems were compared to the effects on genomic DNA replication in BSC-1 cells. Overall, the results support the concept that BLM-induced inhibition of DNA replication occurs by both trans- (inhibition of replication of nondamaged template) and cis-acting mechanisms (template damage).     

Replication of SV40 chromatin in extracts from eggs of Xenopus laevis.

Simian virus 40 (SV40) nucleoprotein complexes were prepared from lytically infected cells and used as primer-templates for DNA replication in protein extracts from Xenopus eggs. We found that nucleoprotein containing replicating SV40 DNA served as primer-template while nucleoprotein with nonreplicating SV40 DNA was ineffective. In vitro DNA synthesis begins with short DNA fragments ("Okazaki fragments") which are, in later steps, joined to give unit length SV40 DNA strands, suggesting that in vivo initiated rounds of replication are completed in vitro in the Xenopus system. This conclusion is supported by a restriction enzyme analysis showing that in vitro DNA synthesis occurs in fragments distal to the SV40 origin of replication. Our studies indicate that SV40 DNA replication in Xenopus extracts can be used an an experimental system to study the biochemistry of replicative DNA chain elongation in vitro.     

cdc2 family kinases phosphorylate a human cell DNA replication factor, RPA, and activate DNA replication.

RPA is a single-stranded DNA binding protein complex purified from human cells and is essential for the initiation and elongation stages of SV40 DNA replication in vitro. In both human and yeast cells, the 34 kDa polypeptide subunit of RPA is phosphorylated in the S and G2 phases of the cell cycle and not in G1. One of the major RPA kinases present in extracts of human cells was purified and shown to be the cyclin B-cdc2 complex. This purified kinase, and a closely related cyclin A associated cdc2-like kinase, phosphorylated RPA p34 on a subset of the chymotryptic peptides that were phosphorylated in vivo at the G1-S transition. Two serines near the N-terminus of RPA p34 were identified as possible sites of phosphorylation by cdc2 kinase. These same serines were necessary for RPA phosphorylation in vivo. The purified cdc2 kinase stimulated SV40 DNA replication in vitro when added to G1 cell extracts. The kinase also stimulated unwinding at the origin of replication, one of the earliest steps in DNA replication requiring RPA, but only in the presence of an additional factor present in G1 cell extracts. Thus, one or more members of the cyclin-cdc2 kinase family may be required for the initiation and maintenance of S phase, in part due to their ability to phosphorylate and activate a cellular DNA replication factor, RPA.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.     

SV40 DNA replication inhibition by the monofunctional DNA alkylator Et743

Ecteinascidin 743 (Et743) is a highly cytotoxic anticancer agent isolated from the squirt Ecteinascidia turbinate, which alkylates DNA in the minor groove at GC-rich sequences resulting in an unusual bending toward the major groove. The ability of Et743 to block DNA replication was studied using the well-established simian virus (SV40) model for mammalian DNA replication in cells and cell-free extracts. Intracellular SV40 DNA isolated from Et743-treated BSC-1 cells was analyzed by native, two-dimensional agarose gel electrophoresis. A low frequency of Et743 adducts detected at 30-100 nM drug concentrations inhibited SV40 origin activity and induced formation of unusual DNA replication intermediates. Under cell-free conditions, only a high Et743 adduct frequency reduced SV40 DNA synthesis. Comparative studies involving related DNA alkylators, tomamycin and saframycin A, revealed inhibition of SV40 DNA replication in cells at concentrations approximately 10 times higher than Et743. Under cell-free conditions tomamycin- or saframycin-A-adducted DNA templates inhibited DNA synthesis similarly to Et743. Et743 appears to be unusual among other alkylators, because its adducts strongly inhibit intracellular SV40 DNA replication but are relatively weak as cis inhibitors as measured under cell-free conditions.     

Effect of ICRF-193, a novel DNA topoisomerase II inhibitor, on simian virus 40 DNA and chromosome replication in vitro.

The effect of ICRF-193, a noncleavable-complex-forming topoisomerase II inhibitor, on simian virus 40 (SV40) DNA and SV40 chromosome replication was examined by using an in vitro replication system composed of HeLa cell extracts and SV40 T antigen. Unlike the topoisomerase inhibitors VP-16 and camptothecin, ICRF-193 had little effect on DNA chain elongation during SV40 DNA replication, but high-molecular-weight DNAs instead of segregated monomer DNAs accumulated as major products. Analysis of the high-molecular-weight DNAs by two-dimensional gel electrophoresis revealed that they consisted of catenated dimers and late Cairns-type DNAs. Incubation of the replicated DNA with topoisomerase II resulted in conversion of the catenated dimers to monomer DNAs. These results indicate that ICRF-193 induces accumulation of catenated dimers and late Cairns-type DNAs by blocking the decatenating and relaxing activities of topoisomerase II in the late stage of SV40 DNA replication. In contrast, DNA replication of SV40 chromosomes was severely blocked by ICRF-193 at the late stage, and no catenated dimers were synthesized. These results are consistent with the finding that topoisomerase II is required for unwinding of the final duplex DNA in the late stage of SV40 chromosome replication in vitro.     

Induction of DNA replication-mediated double strand breaks by psoralen DNA interstrand cross-links

The effect of DNA interstrand cross-links (cross-links) on DNA replication was examined with a cell-free SV40 origin-dependent DNA replication system. A defined template DNA with a single psoralen cross-link and the SV40 origin of replication was replicated by HeLa cell-free extract in the presence of SV40 large T antigen. The psoralen cross-link inhibited DNA replication by terminating chain elongation at 1-50 nucleotides before the cross-linked sites. The termination of DNA replication by the cross-links mediated the generation of double strand breaks near the cross-linked sites. These results are the first biochemical evidence of the generation of double strand breaks by DNA replication.     

Identification of multiple cellular factors required for SV40 replication in vitro

The replication of simian virus 40 has been studied by using cell-free extracts derived from human 293 cells. Fractionation of this extract has led to the identification of three fractions that are required for efficient DNA synthesis. Initial fractionation of the crude extract by phosphocellulose chromatography has produced two fractions, I and II, neither of which is able to support replication separately, but when they are combined, efficient synthesis is restored. Both fractions are required, with SV40 T antigen, for the formation of a presynthesis complex at the SV40 origin. The major replication enzymes, DNA polymerase, DNA primase and the topoisomerases I and II all reside in fraction II. Fraction I has been subdivided into two subfractions (A and B) by DEAE-cellulose chromatography. Fraction A is essential for replication and is required for presynthesis complex formation. Fraction B stimulates DNA replication and is only required at the elongation stage. This multicomponent system has provided the foundation for identification of individual components that are required for DNA replication in vitro.     

Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro.

Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.     

Effects of VM26, a specific inhibitor of type II DNA topoisomerase, on SV40 chromatin replication in vitro.

We have examined the influence of VM26 (teniposide), a specific inhibitor of mammalian type II DNA topoisomerase, on the replication of SV40 minichromosomes in vitro. The replication system we used consists of replicative intermediate SV40 chromatin as substrate which is converted to mature SV40 chromatin in the presence of ATP, deoxynucleotides and a protein extract from uninfected cells. The addition of 100 microM VM26 to this system reduces DNA synthesis to 70 to 80 percent of the control and leads to an accumulation of 'late replicative intermediates'. The VM26 induced block of replication was not released by the addition of large quantities of type I DNA topoisomerase. We conclude, that type II DNA topoisomerase is essential for the final replication steps leading from late Cairns structures of replicative intermediates to monomeric minichromosomes. It appears that type I DNA topoisomerase can function as a swivelase during most of the replicative elongation phase, but must later be replaced by type II DNA topoisomerase.     

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.     

Species-specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA.

Physical and functional interactions of simian virus 40 (SV40) and polyomavirus large-T antigens with DNA polymerase alpha-primase were analyzed to elucidate the molecular basis for the species specificity of polymerase alpha-primase in viral DNA replication. SV40 T antigen associated more efficiently with polymerase alpha-primase in crude human extracts than in mouse extracts, while polyomavirus T antigen interacted preferentially with polymerase alpha-primase in mouse extracts. The apparent species specificity of complex formation was not observed when purified polymerase alpha-primases were substituted for the crude extracts. Several functional interactions between T antigen and purified polymerase alpha-primase, including stimulation of primer synthesis and primer elongation on M13 DNA in the presence or absence of the single-stranded DNA binding protein RP-A, also proved to be independent of the species from which polymerase alpha-primase had been purified. However, the human DNA polymerase alpha-primase was specifically required for primosome assembly and primer synthesis on SV40 origin DNA in the presence of T antigen and RP-A.     

Region-specific DNA damage by AT-specific DNA-reactive drugs is predicted by drug binding specificity

Bizelesin and adozelesin are DNA-reactive antitumor drugs that alkylate adenines at the 3' ends of their preferred binding sites [5'T(A/T)(4)A3'and 5'(A/T)(3)(-4)A3', respectively]. We used these drugs to examine the determinants for region-specific damage of human genomic DNA. The distribution of bizelesin binding motifs in several regions analyzed "in silico" correlated well with the experimentally determined lesions in these regions assessed by quantitative polymerase chain reaction (QPCR) stop assay. In contrast to the typically low motif density, clusters of potential bizelesin binding sites were found in the matrix-associated regions (MAR domains) of the c-myc and apolipoprotein B (apoB) genes. Accordingly, lesions induced by bizelesin in these domains (2.13 and 7.06 lesions kbp(-1) microM(-1), respectively) markedly exceeded lesions in bulk DNA (0.87 lesions kbp(-1) microM(-1)) or in regions with typically low motif density (e.g., 0.75 and 0.87 lesions kbp(-1) microM(-1) in a beta-globin gene and c-myc origin of replication regions, respectively). Consistent with the more frequent, less localized adozelesin motif, actual lesions induced by adozelesin exceeded by severalfold lesions by bizelesin in four selected regions (within the c-myc and HPRT loci). Whereas adozelesin is likely to affect similar regions as bizelesin, adozelesin's more promiscuous binding probably compromises its relative specificity for such targets. In contrast, findings for bizelesin provide for the first time a proof of principle that a small molecular weight drug can preferentially damage specific regions in cellular DNA. Targeting of critical repetitive sequences, such as AT-rich MAR domains, which allow for clustering of drug binding motif, can be the paradigm for region specificity of small molecular weight agents.     

Effect of the antitumor drug cis-diamminedichloroplatinum(II) and related platinum complexes on eukaryotic DNA replication

An SV40-based in vitro replication system has been used to examine the effects of platinum compounds on eukaryotic DNA replication. Plasmid templates containing the SV40 origin of replication were modified with the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) or the inactive analogues [Pt(dien)Cl]+ and trans-DDP. The platinated plasmids were used as templates for DNA synthesis by the DNA polymerases present in cytosolic extracts prepared from human cell lines HeLa and 293. Bifunctional adducts formed by cis- and trans-DDP inhibited DNA replication by 95% at a bound drug to nucleotide ratio [(D/N)b] of less than 9 x 10(-4), in contrast to the monofunctional [Pt(dien)Cl]+ analogues, which required a (D/N)b of 3.4 x 10(-3) for 62% inhibition of DNA replication. An average of two platinum adducts per genome was sufficient for inhibition of DNA replication by cisplatin. When trans-DDP-modified, but not cis-DDP-modified, SV40 origin containing plasmids [(D/N)b = 1.7 x 10(-3)] were allowed to incubate in the 293 cytosolic extracts for 1 h prior to addition of T-antigen to initiate replication, DNA synthesis was restored to 30% of control. This result suggested the presence of an activity in the extracts that reactivates trans-DDP-modified DNA templates for replication. This hypothesis was confirmed by an in vitro nucleotide excision repair assay that revealed activity in 293 and HeLa cell extracts selective for trans-DDP-modified plasmid DNAs. Such selective repair of trans-DDP-damaged DNA in human cells would contribute to its lack of antitumor activity.     

Regulation of DNA replication by homopurine/homopyrimidine sequences

The simple repeating homopurine/homopyrimidine sequences dispersed throughout many eukaryotic genomes are known to form triple helical structures comprising three-stranded and single-stranded DNA. Several lines of evidence suggest that these structures influence DNA replication in cells. Homopurine/homopyrimidine sequences cloned into simian virus 40 (SV40) or SV40 origin-containing plasmids caused a reduced rate of DNA synthesis due to the pausing of replication forks. More prominent arrests were observed in in vitro experiments using single-stranded and double-stranded DNA with triplex-forming sequences. Nucleotides unable to form triplexes when present in the template DNA or when incorporated into the nascent strand prevented termination. Similarly, mutations destroying the triplex potential did not cause arrest while compensatory mutations restoring triplex potential restored it. These and other observations from a number of laboratories indicating that homopurine/homopyrimidine sequences act as arrest signals in vitro and as pause sites in vivo during replication fork movement suggest that these naturally occurring sequences play a regulatory role in DNA replication and gene amplification.     

Effects of VM26 (teniposide), a specific inhibitor of type II DNA topoisomerase, on SV40 DNA replication in vivo.

Simian Virus 40 (SV40) infected cells were pulse labeled with (3H) thymidine and chased either in the absence or in the presence of the cytotoxic drug VM26 (teniposide). We investigated the structure of labeled SV40 DNA and found that VM26 had no significant effect on replicative chain elongation but strongly inhibited the conversion of late replication intermediates to mature DNA daughter molecules. The late replicative SV40 DNA intermediates which accumulate in VM26 treated cells contained essentially full length labeled DNA strands. These newly synthesized strands were not part of two catenated interlocked SV40 monomers suggesting that the block occurred prior to the final ligation reaction. Since VM26 is known to be a specific inhibitor of DNA topoisomerase II we conclude that this enzyme is dispensable for the chain elongation of replicating SV40 DNA, but that it is essential for the termination of SV40 DNA replication cycles.     

Simian virus 40 DNA replication in vitro: specificity of initiation and evidence for bidirectional replication.

We recently described a soluble cell-free system derived from monkey cells that is capable of replicating exogenous plasmid DNA molecules containing the simian virus 40 (SV40) origin of replication (J.J. Li, and T.J. Kelly, Proc. Natl. Acad. Sci. U.S.A. 81:6973-6977, 1984). Replication in the system is completely dependent upon the addition of the SV40 large T antigen. In this report we describe additional properties of the in vitro replication reaction. Extracts prepared from cells of several nonsimian species were tested for the ability to support origin-dependent replication in the presence of T antigen. The activities of extracts derived from human cell lines HeLa and 293 were approximately the same as those of monkey cell extracts. Chinese hamster ovary cell extracts also supported SV40 DNA replication in vitro, but the extent of replication was approximately 1% of that observed with human or monkey cell extracts. No replication activity was detectable in extracts derived from BALB/3T3 mouse cells. The ability of these extracts to support replication in vitro closely parallels the ability of the same cells to support replication in vivo. We also examined the ability of various DNA molecules containing sequences homologous to the SV40 origin to serve as templates in the cell-free system. Plasmids containing the origins of human papovaviruses BKV and JCV replicated with an efficiency 10 to 20% of that of plasmids containing the SV40 origin. Plasmids containing Alu repeat sequences (BLUR8) did not support detectable DNA replication in vitro. Circular DNA molecules were found to be the best templates for DNA replication in the cell-free system; however, linear DNA molecules containing the SV40 origin also replicated to a significant extent (10 to 20% of circular molecules). Finally, electron microscopy of replication intermediates demonstrated that the initiation of DNA synthesis in vivo takes place at a unique site corresponding to the in vivo origin and that replication is bidirectional. These findings provide further evidence that replication in the cell-free system faithfully mimics SV40 DNA replication in vivo.