Ubiquitylation of proliferating cell nuclear antigen and recruitment of human DNA polymerase eta

This study investigated the requirement for ubiquitylation of PCNA at lysine 164 during polymerase eta-dependent translesion synthesis (TLS) of site-specific cis-syn cyclobutane thymine dimers (T (wedge)T). The in vitro assay recapitulated origin-dependent initiation, fork assembly, and semiconservative, bidirectional replication of double-stranded circular DNA substrates. A phosphocellulose column was used to fractionate HeLa cell extracts into two fractions; flow-through column fraction I (CFI) contained endogenous PCNA, RPA, ubiquitin-activating enzyme E1, and ubiquitin conjugase Rad6, and eluted column fraction II (CFII) included pol delta, pol eta, and RFC. CFII supplemented with purified recombinant RPA and PCNA (wild type or K164R, in which lysine was replaced with arginine) was competent for DNA replication and TLS. K164R-PCNA complemented CFII for these activities to the same extent and efficiency as wild-type PCNA. CFII mixed with CFI (endogenous PCNA, E1, Rad6) exhibited enhanced DNA replication activity, but the same TLS efficiency determined with the purified proteins. These results demonstrate that PCNA ubiquitylation at K164 of PCNA is not required in vitro for pol eta to gain access to replication complexes at forks stalled by T (wedge)T and to catalyze TLS across this dimer.     

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.     

Purification of DNA polymerase delta as an essential simian virus 40 DNA replication factor.

DNA replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.     

The human stress-activated protein kin17 belongs to the multiprotein DNA replication complex and associates in vivo with mammalian replication origins

The human stress-activated protein kin17 accumulates in the nuclei of proliferating cells with predominant colocalization with sites of active DNA replication. The distribution of kin17 protein is in equilibrium between chromatin-DNA and the nuclear matrix. An increased association with nonchromatin nuclear structure is observed in S-phase cells. We demonstrated here that kin17 protein strongly associates in vivo with DNA fragments containing replication origins in both human HeLa and monkey CV-1 cells. This association was 10-fold higher than that observed with nonorigin control DNA fragments in exponentially growing cells. In addition, the association of kin17 protein to DNA fragments containing replication origins was also analyzed as a function of the cell cycle. High binding of kin17 protein was found at the G(1)/S border and throughout the S phase and was negligible in both G(0) and M phases. Specific monoclonal antibodies against kin17 protein induced a threefold inhibition of in vitro DNA replication of a plasmid containing a minimal replication origin that could be partially restored by the addition of recombinant kin17 protein. Immunoelectron microscopy confirmed the colocalization of kin17 protein with replication proteins like RPA, PCNA, and DNA polymerase alpha. A two-step chromatographic fractionation of nuclear extracts from HeLa cells revealed that kin17 protein localized in vivo in distinct protein complexes of high molecular weight. We found that kin17 protein purified within an approximately 600-kDa protein complex able to support in vitro DNA replication by means of two different biochemical methods designed to isolate replication complexes. In addition, the reduced in vitro DNA replication activity of the multiprotein replication complex after immunodepletion for kin17 protein highlighted for a direct role in DNA replication at the origins.     

Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells.

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.     

Purification of host cell enzymes involved in adeno-associated virus DNA replication

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase delta, but not polymerase epsilon or alpha, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase delta complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase delta, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase delta, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.     

Isolation of a stimulatory factor for nuclear DNA replication

Aqueous extracts of isolated nuclei and intact plasmodia of Physarum contain a heat-stable stimulator of nuclear DNA replication. The stimulatory factor is present throughout the mitotic cycle, and its activity is unaffected by prior exposure of plasmodia to cycloheximide. The stimulatory substance has been partially purified by heat treatment, precipitation with ethanol, chromatography on DEAE cellulose, and gel filtration. The purified material contains both carbohydrate and protein, and exhibits a molecular weight of about 30 000. The active substance increases the rate and overall extent of DNA replication in S-phase nuclei, but does not trigger the initiation of DNA synthesis in nuclei isolated from G2-phase plasmodia. The stimulatory material contains little or no deoxyribonuclease or DNA polymerase activity, and it does not affect DNA polymerase activity assayed using a purified DNA template.     

Human mismatch repair: reconstitution of a nick-directed bidirectional reaction

Bidirectional mismatch repair directed by a strand break located 3' or 5' to the mispair has been reconstituted using seven purified human activities: MutSalpha, MutLalpha, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase delta. In addition to DNA polymerase delta, PCNA, RFC, and RPA, 5'-directed repair depends on MutSalpha and EXOI, whereas 3'-directed mismatch correction also requires MutLalpha. The repair reaction displays specificity for DNA polymerase delta, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase delta in the excision step of repair. RFC and PCNA dramatically activate polymerase delta-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase delta are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3'- and 5'-directed excision in HeLa nuclear extracts by only 20-30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase.     

Molecular cloning, structure and expression of the yeast proliferating cell nuclear antigen gene.

The budding yeast Saccharomyces cerevisiae is proving to be an useful and accurate model for eukaryotic DNA replication. It contains both DNA polymerase alpha (I) and delta (III). Recently, proliferating cell nuclear antigen (PCNA), which in mammalian cells is an auxiliary subunit of DNA polymerase delta and is essential for in vitro leading strand SV40 DNA replication, was purified from yeast. We have now cloned the gene for yeast PCNA (POL30). The gene codes for an essential protein of 29 kDa, which shows 35% homology with human PCNA. Cell cycle expression studies, using synchronized cells, show that expression of both the PCNA (POL30) and the DNA polymerase delta (POL3, or CDC2) genes of yeast are regulated in an identical fashion to that of the DNA polymerase alpha (POL1) gene. Thus, steady state mRNA levels increase 10-100-fold in late G1 phase, peak in early S-phase, and decrease to low levels in late S-phase. In addition, in meiosis mRNA levels increase prior to initiation of premeiotic DNA synthesis.     

Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.

Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase.     

DNA replication in isolated HeLa cell nuclei

DNA replication was investigated in HeLa cell nuclei isolated from different phases of the cell cycle. DNA synthesis occurred only in S-phase nuclei and was dependent on the presence of the four deoxynucleoside triphosphates, Mg⁺⁺, ATP and S-phase cytoplasm. G₁-phase cytoplasm was unable to support such DNA synthesis. A purified preparation of calf thymus DNA polymerase, however, was able to replace S-phase cytoplasm in supporting ATP dependent DNA synthesis, which suggests that the S-phase cytoplasmic factor is a DNA polymerase. G₁-phase nuclei could under no conditions be stimulated to initiate DNA replication prematurely.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication mitiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Stability and nuclear distribution of mammalian replication protein A heterotrimeric complex

Replication protein A (RPA), a stable complex of three polypeptides, is the single-stranded DNA-binding protein essential for DNA replication in eukaryotic cells. Previous studies of the subcellular distribution and stability of the RPA heterotrimer during the mammalian cell cycle have produced conflicting results. Here, we present evidence that these inconsistencies can be accounted for by the presence of an extractable pool of soluble RPA within the nucleus. Indirect immunofluorescence experiments in both CHO and HeLa cells showed that all three RPA subunits associated specifically with sites of ongoing DNA synthesis, similar to the replication fork protein proliferating cell nuclear antigen. Furthermore, we found no evidence for disassembly of the chromatin-bound heterotrimeric RPA complex in vivo. Our results are consistent with a role for RPA in the initiation and elongation steps of replication, as previously defined in the viral in vitro replication systems.     

Cellular factors required for papillomavirus DNA replication.

In vitro replication of papillomavirus DNA has been carried out with a combination of purified proteins and partially purified extracts made from human cells. DNA synthesis requires the viral E1 protein and the papillomavirus origin of replication. The E2 protein stimulates DNA synthesis in a binding site-independent manner. Papillomavirus DNA replication is also dependent on the cellular factors replication protein A, replication factor C, and proliferating-cell nuclear antigen as well as a phosphocellulose column fraction (IIA). Fraction IIA contains DNA polymerase alpha-primase and DNA polymerase delta. Both of these polymerases are essential for papillomavirus DNA replication in vitro. However, unlike the case with T-antigen-dependent replication from the simian virus 40 origin, purified DNA polymerase alpha-primase and delta cannot efficiently replace fraction IIA in the replication reaction. Hence, additional cellular factors seem to be required for papillomavirus DNA replication. Interestingly, replication factor C and proliferating-cell nuclear antigen are more stringently required for DNA synthesis in the papillomavirus system than in the simian virus 40 in vitro system. These distinctions indicate that there must be mechanistic differences between the DNA replication systems of papillomavirus and simian virus 40.     

Mediation of proliferating cell nuclear antigen (PCNA)-dependent DNA replication through a conserved p21(Cip1)-like PCNA-binding motif present in the third subunit of human DNA polymerase delta

The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human DNA polymerase delta has not been clearly defined. We show that the third subunit of human DNA polymerase delta, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previously shown to be important for DNA polymerase delta activity and for binding of the cell cycle inhibitor p21(Cip1). In accordance with this, a peptide containing the PCNA-binding domain of p21(Cip1) inhibited p66 binding to PCNA and the activity of native three-subunit DNA polymerase delta. Furthermore, pull-down assays showed that DNA polymerase delta requires p66 for interaction with PCNA. More importantly, only reconstituted three-subunit DNA polymerase delta displayed PCNA-dependent DNA replication that could be inhibited by the PCNA-binding domain of p21(Cip1). Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co-localization of p66 with PCNA and DNA polymerase delta within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle-dependent regulation. These results suggest that p66 is the chief mediator of PCNA-dependent DNA synthesis by DNA polymerase delta.     

Characterization of an improved in vitro DNA replication system for Escherichia coli plasmids.

A modified in vitro replication system has been characterized and used to catalogue the host proteins required for the replication of plasmid RSF1030. These extracts differ from systems described previously in that endogenous DNA is removed. Replication in vitro therefore requires an exogenouos RSF1030. Synthesis in the in vitro system faithfully mimics in vivo replication with respect to the products synthesized, effects of specific inhibitors, and requirements for RNA polymerase and DNA polymerase I. In addition, we find that proteins encoded by dnaB, dnaC, dnaG, dnaI, dnaP and polC (DNA polymerase III), are required for in vitro plasmid synthesis. The product of dnaA is not required. Extracts prepared from E. coli mutants deficient in in vitro replication can be complemented by addition of purified proteins or of extracts carrying the wild type protein.     

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.     

Detection of a bromoperoxidase in Streptomyces phaeochromogenes

Adenovirus DNA replication was studied in a partially reconstituted system consisting of purified viral proteins (DNA-binding protein, precursor terminal protein and Ad DNA polymerase) and a nuclear extract from uninfected HeLa cells. Optimal DNA replication required the presence of a heat-stable, ribonuclease-sensitive fraction from the cytosol of uninfected cells. This fraction stimulated the initiation about 3-fold and the replication of origin fragments 5-10-fold. Sedimentation analysis indicated the presence of a fast-sedimenting and a slow-sedimenting component which complemented each other. At least part of the stimulation was caused by low-molecular-mass RNA.     

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication

We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC₅₀ of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser¹³⁹)histone H2AX induction and cell growth inhibition was observed.     

DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum

Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.