The midblastula transition defines the onset of Y RNA-dependent DNA replication in Xenopus laevis

Noncoding Y RNAs are essential for the initiation of chromosomal DNA replication in mammalian cell extracts, but their role in this process during early vertebrate development is unknown. Here, we use antisense morpholino nucleotides (MOs) to investigate Y RNA function in Xenopus laevis and zebrafish embryos. We show that embryos in which Y RNA function is inhibited by MOs develop normally until the midblastula transition (MBT) but then fail to replicate their DNA and die before gastrulation. Consistent with this observation, Y RNA function is not required for DNA replication in Xenopus egg extracts but is required for replication in a post-MBT cell line. Y RNAs do not bind chromatin in karyomeres before MBT, but they associate with interphase nuclei after MBT in an origin recognition complex (ORC)-dependent manner. Y RNA-specific MOs inhibit the association of Y RNAs with ORC, Cdt1, and HMGA1a proteins, suggesting that these molecular associations are essential for Y RNA function in DNA replication. The MBT is thus a transition point between Y RNA-independent and Y RNA-dependent control of vertebrate DNA replication. Our data suggest that in vertebrates Y RNAs function as a developmentally regulated layer of control over the evolutionarily conserved eukaryotic DNA replication machinery.     

In vitro replication of adeno-associated virus DNA: enhancement by extracts from adenovirus-infected HeLa cells.

Previously we have described an in vitro assay for the replication of adeno-associated virus type 2 (AAV2) DNA. Addition of the AAV2 nonstructural protein Rep68 to an extract from uninfected cells supports the replication of linear duplex AAV DNA. In this report, we examine replication of linear duplex AAV DNA in extracts from either uninfected or adenovirus (Ad)-infected HeLa cells. The incorporation of radiolabeled nucleotides into full-length linear AAV DNA is 50-fold greater in extracts from Ad-infected cells than in extracts from uninfected cells. In addition, the majority of the labeled full-length AAV DNA molecules synthesized in the Ad-infected extract have two newly replicated strands, whereas the majority of labeled full-length AAV DNA molecules synthesized in the uninfected extract have only one newly replicated strand. The numbers of replication initiations on original templates in the two assays are approximately the same; however, replication in the case of the Ad-infected cell extract is much more likely to result in the synthesis of a full-length AAV DNA molecule. Most of the newly replicated molecules in the assay using uninfected cell extracts are in the form of stem-loop structures. We hypothesize that Ad infection provides a helper function related to elongation during replication by a single-strand displacement mechanism. In the assay using the uninfected HeLa cell extract, replication frequently stalls before reaching the end of the genome, causing the newly synthesized strand to be displaced from the template, with a consequent folding on itself and replication back through the inverted terminal repeat, using itself as a template. In support of this conjecture, replication in the uninfected cell extract of shorter substrate molecules is more efficient, as measured by incorporation of radiolabeled nucleotides into full-length substrate DNA. In addition, when shorter substrate molecules are used as the template in the uninfected HeLa cell assay, a greater proportion of the labeled full-length substrate molecules contain two newly replicated strands. Shorter substrate molecules have no replicative advantage over full-length substrate molecules in the assay using an extract from Ad-infected cells.     

Heteroduplex repair in extracts of human HeLa cells

A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.     

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.     

Plasmids bearing mammalian DNA-replication origin-enriched (ors) fragments initiate semiconservative replication in a cell-free system.

Four plasmids containing monkey (CV-1) origin-enriched sequences (ors), which we have previously shown to replicate autonomously in CV-1, COS-7 and HeLa cells (Frappier and Zannis-Hadjopoulos (1987) Proc. Natl. Acad. Sci. USA 84, 6668-6672), were found to replicate in an in vitro replication system using HeLa cell extracts. De novo site-specific initiation of replication on plasmids required the presence of an ors sequence, soluble low-salt cytosolic extract, poly(ethylene glycol), a solution containing the four standard deoxyribonucleoside triphosphates and an ATP regenerating system. The major reaction products migrated as relaxed circular and linear plasmid DNAs, both in the presence and absence of high-salt nuclear extracts. Inclusion of high-salt nuclear extract was required to obtain closed circular supercoiled molecules. Replicative intermediates migrating slower than form II and topoisomers migrating between forms II and I were also included among the replication products. Replication of the ors plasmids was not inhibited by ddTTP, an inhibitor of DNA polymerase beta and gamma, and was sensitive to aphidicolin indicating that DNA polymerase alpha and/or delta was responsible for DNA synthesis. Origin mapping experiments showed that early in the in vitro replication reaction, incorporation of nucleotides occurs preferentially at ors-containing fragments, indicating ors specific initiation of replication. In contrast, the limited incorporation of nucleotides into pBR322, was not site specific. The observed synthesis was semiconservative and appeared to be bidirectional.     

In vitro (CTG)*(CAG) expansions and deletions by human cell extracts

The mechanism of disease-associated (CTG)*(CAG) expansion may involve DNA replication slippage, replication direction, Okazaki fragment processing, recombination, or repair. A length-dependent bias for expansions is observed in humans affected by a trinucleotide repeat-associated disease. We developed an assay to test the effect of replication direction on (CTG)*(CAG) instabilities incurred during in vitro (SV40) DNA replication mediated by human cell extracts. This system recapitulates the bias for expansions observed in humans. Replication by HeLa cell extracts generated expansions and deletions that depended upon repeat tract length and the direction of replication. Templates with 79 repeats yielded predominantly expansions (CAG as lagging strand template) or predominantly deletions (CTG as lagging strand template). Templates containing 17 repeats were stable. Thus, replication direction determined the type of mutation. These results provide new insights into the orientation of replication effect upon repeat stability. This system will be useful in determining the contribution of specific human proteins to (CTG)*(CAG) expansions.     

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.     

Receptor independent effects on DNA replication by steroids

There is now convincing evidence associating estrogens with an increased risk of some cancers. However, the absence of a complete correlation between estrogen receptor binding and the biological activity of these estrogens has suggested the possibility of other mechanisms of action. The effect on DNA replication of several hormones that are putatively involved in breast cancer was tested at a physiological concentration. The studies were conducted in a HeLa cell-free system by using a plasmid containing a specific mammalian origin of replication (DHFR oriβ<0R) as template DNA. A series of related steroids produced an entire range of activity from enhancement to inhibition of in vitro DNA replication. These studies indicate a new possible target, which may help to better understand the effect of these hormones in breast cancer. Furthermore, the results show that this in vitro DNA replication system provides an evaluative assay for the effects of compounds on hormone-responsive cancers independent of some hormone receptors. J. Cell. Biochem. 70:323–329, 1998. © 1998 Wiley-Liss, Inc.     

UV light-induced DNA synthesis arrest in HeLa cells is associated with changes in phosphorylation of human single-stranded DNA-binding protein.

We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV-induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single-stranded DNA-binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV-irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV-induced DNA synthesis arrest may be mediated in part through phosphorylation-related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.     

DNA excision repair in mammalian cell extracts.

The many genetic complementation groups of DNA excision-repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell extracts and is defective in extracts from cell lines derived from individuals with the excision-repair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single-stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication.     

DNA helicase and duplex DNA fragment unwinding activities of polyoma and simian virus 40 large T antigen display similarities and differences.

We have characterized the biochemical activities of purified polyoma (Py) large T antigen (T Ag) that was capable of mediating the replication of a plasmid containing the Py origin (ori(+) DNA) in mouse cell extracts. We report here that like the T Ag encoded by simian virus 40 (SV40), Py T Ag has DNA helicase and double-stranded DNA fragment unwinding activities. Py T Ag displaced DNA fragments greater than 1,600 nucleotides which were annealed to complementary sequences in single-stranded M13 by translocating in the 3' to 5' direction. Both helicase and double-stranded DNA fragment unwinding reactions were completely dependent upon NTP hydrolysis, displaying a strong preference for ATP and dATP. At low T Ag concentrations, significantly more Py ori(+) DNA fragment was unwound compared with a fragment lacking the replication origin. However, at higher ratios of Py T Ag to DNA, equivalent to those used in replication reactions, unwinding of both ori-containing and -lacking fragments was equally efficient. This is in contrast to SV40 T Ag which exhibited a more stringent requirement for SV40 origin sequences under similar conditions. Furthermore, some of the nucleotides that supported the helicase and unwinding activities of Py T Ag were different from those for the same SV40 T Ag reactions. We have also observed that in contrast to the very poor replication of linear SV40 ori(+) DNA by SV40 T Ag in human cell extracts, linear Py ori(+) DNA was replicated efficiently in mouse cell extracts by Py T Ag. However, despite the fact that linear Py ori(+), SV40 ori(+), and ori(-) DNA fragments could be unwound with comparable efficiency by Py T Ag, only fragments containing the Py replication origin were replicated in vitro. These results suggest that the initiation of DNA synthesis at the Py origin of replication requires features in addition to unwinding of the template.     

Cofractionation of hela cell replication proteins with Ors-binding activity

Ors (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV-1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE-Sephadex and then on a Affi-Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band-shift elution experiments of the specific protein-DNA complex detect by silver-staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases α and δ, topoisomerase II, and replication protein A, (RP-A).     

Butylanilinouracil: a selective inhibitor of HeLa cell DNA synthesis and HeLa cell DNA polymerase alpha.

A series of 6-anilinouracils, dGTP analogues which selectively inhibit specific bacterial DNA polymerases, were examined for their capacity to inhibit purified DNA polymerases from HeLa cells. The p-n-butyl derivative (BuAU) was found to inhibit DNA polymerase alpha with a Ki of approximately 60 microM. The inhibitory effect of BuAU was reversed specifically by dGTP and was observed only for DNA polymerase alpha; polymerases beta and lambda were not inhibited by drug at concentrations as high as 1 mM. BuAU also was inhibitory in vivo in HeLa cell culture; at 100 microM it reversibly inhibited cell division and selectively depressed DNA synthesis. The results of these studies indicate that BuAU is an inhibitor with considerable potential as a specific probe with which to dissect the structure of mammalian polymerase alpha and its putative role in cellular DNA replication.     

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.     

Purification of a cellular, double-stranded DNA-binding protein required for initiation of adenovirus DNA replication by using a rapid filter-binding assay.

A rapid and quantitative nitrocellulose filter-binding assay is described for the detection of nuclear factor I, a HeLa cell sequence-specific DNA-binding protein required for the initiation of adenovirus DNA replication. In this assay, the abundant nonspecific DNA-binding activity present in unfractionated HeLa nuclear extracts was greatly reduced by preincubation of these extracts with a homopolymeric competitor DNA. Subsequently, specific DNA-binding activity was detected as the preferential retention of a labeled 48-base-pair DNA fragment containing a functional nuclear factor I binding site compared with a control DNA fragment to which nuclear factor I did not bind specifically. This specific DNA-binding activity was shown to be both quantitative and time dependent. Furthermore, the conditions of this assay allowed footprinting of nuclear factor I in unfractionated HeLa nuclear extracts and quantitative detection of the protein during purification. Using unfrozen HeLa cells and reagents known to limit endogenous proteolysis, nuclear factor I was purified to near homogeneity from HeLa nuclear extracts by a combination of standard chromatography and specific DNA affinity chromatography. Over a 400-fold purification of nuclear factor I, on the basis of the specific activity of both sequence-specific DNA binding and complementation of adenovirus DNA replication in vitro, was affected by this purification. The most highly purified fraction was greatly enriched for a polypeptide of 160 kilodaltons on silver-stained sodium dodecyl sulfate-polyacrylamide gels. Furthermore, this protein cosedimented with specific DNA-binding activity on glycerol gradients. That this fraction indeed contained nuclear factor I was demonstrated by both DNase I footprinting and its function in the initiation of adenovirus DNA replication. Finally, the stoichiometry of specific DNA binding by nuclear factor I is shown to be most consistent with 2 mol of the 160-kilodalton polypeptide binding per mol of nuclear factor I-binding site.     

Reversible protein phosphorylation modulates nucleotide excision repair of damaged DNA by human cell extracts.

Nucleotide excision repair of DNA in mammalian cells uses more than 20 polypeptides to remove DNA lesions caused by UV light and other mutagens. To investigate whether reversible protein phosphorylation can significantly modulate this repair mechanism we studied the effect of specific inhibitors of Ser/Thr protein phosphatases. The ability of HeLa cell extracts to carry out nucleotide excision repair in vitro was highly sensitive to three toxins (okadaic acid, microcystin-LR and tautomycin), which block PP1- and PP2A-type phosphatases. Repair was more sensitive to okadaic acid than to tautomycin, suggesting the involvement of a PP2A-type enzyme, and was insensitive to inhibitor-2, which exclusively inhibits PP1-type enzymes. In a repair synthesis assay the toxins gave 70% inhibition of activity. Full activity could be restored to toxin-inhibited extracts by addition of purified PP2A, but not PP1. The p34 subunit of replication protein A was hyperphosphorylated in cell extracts in the presence of phosphatase inhibitors, but we found no evidence that this affected repair. In a coupled incision/synthesis repair assay okadaic acid decreased the production of incision intermediates in the repair reaction. The formation of 25-30mer oligonucleotides by dual incision during repair was also inhibited by okadaic acid and inhibition could be reversed with PP2A. Thus Ser/Thr- specific protein phosphorylation plays an important role in the modulation of nucleotide excision repair in vitro.     

Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection

We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and , we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.