Distribution of Replicating Simian Virus 40 DNA in Intact Cells and Its Maturation in Isolated Nuclei

The maturation of replicating simian virus 40 (SV40) chromosomes into superhelical viral DNA monomers [SV40(I) DNA] was analyzed in both intact cells and isolated nuclei to investigate further the role of soluble cytosol factors in subcellular systems. Replicating intermediates [SV40(RI) DNA] were purified to avoid contamination by molecules broken at their replication forks, and the distribution of SV40(RI) DNA as a function of its extent of replication was analyzed by gel electrophoresis and electron microscopy. With virus-infected CV-1 cells, SV40(RI) DNA accumulated only when replication was 85 to 95% completed. These molecules [SV40(RI^*) DNA] were two to three times more prevalent than an equivalent sample of early replicating DNA, consistent with a rate-limiting step in the separation of sibling chromosomes. Nuclei isolated from infected cells permitted normal maturation of SV40(RI) DNA into SV40(I) DNA when the preparation was supplemented with cytosol. However, in the absence of cytosol, the extent of DNA synthesis was diminished three- to fivefold (regardless of the addition of ribonucleotide triphosphates), with little change in the rate of synthesis during the first minute; also, the joining of Okazaki fragments to long nascent DNA was inhibited, and SV40(I) DNA was not formed. The fraction of short-nascent DNA chains that may have resulted from dUTP incorporation was insignificant in nuclei with or without cytosol. Pulse-chase experiments revealed that joining, but not initiation, of Okazaki fragments required cytosol. Cessation of DNA synthesis in nuclei without cytosol could be explained by an increased probability for cleavage of replication forks. These broken molecules masqueraded during gel electrophoresis of replicating DNA as a peak of 80% completed SV40(RI) DNA. Failure to convert SV40(RI^*) DNA into SV40(I) DNA under these conditions could be explained by the requirement for cytosol to complete the gap-filling step in Okazaki fragment metabolism: circular monomers with their nascent DNA strands interrupted in the termination region [SV40(II^*) DNA] accumulated with unjoined Okazaki fragments. Thus, separation of sibling chromosomes still occurred, but gaps remained in the terminal portions of their daughter DNA strands. These and other data support a central role for SV40(RI^*) and SV40(II^*) DNAs in the completion of viral DNA replication.     

An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains.

It is generally accepted that an aphidicolin-sensitive DNA polymerase elongates the eucaryotic RNA primer (iRNA) into a mature Okazaki piece reaching ca. 200 nucleotides. Yet, as shown here, nascent DNA chains below 40 nucleotides accumulated in simian virus 40 (SV40) DNA replicating in isolated nuclei in the presence of aphidicolin. These products resembled precursors of longer Okazaki pieces synthesized in the absence of aphidicolin (termed here DNA primers) in size distribution, lagging-replication-fork polarity, and content of iRNA. Within the isolated SV40 replicative intermediate, DNA primers could be extended in a reaction catalyzed by the Escherichia coli DNA polymerase I large fragment. This increased their length by an average of 21 deoxyribonucleotide residues, indicating that single-stranded gaps of corresponding length existed 3' to the DNA primers. Incubation with T4 DNA ligase converted most of the extended DNA primers into products resembling long Okazaki pieces. These data led us to propose that the synthesis of an SV40 Okazaki piece could be itself discontinuous and could comprise the following steps: (i) iRNA synthesis by DNA primase, (ii) iRNA extension into a DNA primer by an aphidicolin-resistant activity associated with DNA primase-DNA polymerase alpha, (iii) removal of iRNA moieties between adjacent DNA primers, (iv) "gap filling" between DNA primers by the aphidicolin-sensitive DNA polymerase alpha, and (v) ligation of DNA primer units onto a growing Okazaki piece. Eventually, a mature Okazaki piece is ligated onto a longer nascent DNA chain.     

RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.     

Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells

Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo

Analysis of the nucleotide sequences at the 5' ends of RNA-primed nascent DNA chains (Okazaki fragments) and of their locations in replicating simian virus 40 (SV40) DNA revealed the precise nature of Okazaki fragment initiation sites in vivo. The primary initiation site for mammalian DNA primase was 3'-purine-dT-5' in the DNA template and the secondary site was 3'-purine-dC-5', with the 5' end of the RNA primer complementary to either the dT or dC. The third position of the initiation site was variable with a preference for dT or dA. About 81% of the available 3'-purine-dT-5' sites and 20% of the 3'-purine-dC-5' sites were used. Purine-rich sites, such as PuPuPu and PyPuPu , were excluded. The 5'-terminal ribonucleotide composition of Okazaki fragments corroborated these conclusions. Furthermore, the length of individual RNA primers was not unique, but varied in size from six to ten bases with some appearing as short as three bases and some as long as 12 bases, depending on the initiation site used. This result was consistent with the average size (9 to 11 bases) of RNA primers isolated from specific regions of the genome. Excision of RNA primers did not appear to stop at the RNA-DNA junction, but removed a variable number of deoxyribonucleotides from the 5' end of the nascent DNA chain. Finally, only one-fourth of the replication forks contained an Okazaki fragment, and the distribution of their initiation sites between the two arms revealed that Okazaki fragments were initiated exclusively (99%) on retrograde DNA templates. The data obtained at two genomic sites about 350 and 1780 bases from ori were essentially the same as that reported for the ori region (Hay & DePamphilis , 1982), suggesting that the mechanism used to synthesize the first DNA chain at ori is the same as that used to synthesize Okazaki fragments throughout the genome.     

Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro.

In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.     

Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized simian virus 40 deoxyribonucleic acid

Exonucleases specific for either 3' ends (Escherichia coli exonuclease III) or 5' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains have been used to determine the number of nucleotides from the actual sites of DNA synthesis to the first nucleosome on each arm of replication forks in simian virus 40 (SV40) chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes, as demonstrated by the following observations: (i) digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period; (ii) in briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains; (iii) the fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA; (iv) the effects of the two exonucleases were additive, consistent with each enzyme trimming only the 3' or 5' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. Accordingly, the average distance from either 3' or 5' ends of long nascent DNA chains to the first nucleosome on either arm of replication forks was found to be 125 nucleotides. Furthermore, each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than one-fifth of the Okazaki fragments were contained in nucleosomes. On the basis of these and other results, a model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. In addition, it is proposed that Okazaki fragments are initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5' ends of long nascent DNA chains is completed.     

Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.     

The incorporation of uracil into animal cell DNA in vitro

In the presence of dUTP, net DNA synthesis in vitro is substantially reduced. Small DNA fragments that arise during in vitro DNA synthesis in the presence of dUTP are produced as a result of dUMP incorporation and subsequent post-replication excision repair process initiated by uracil-DNA-glycosylase. The size of the fragments is dependent upon the amount of dUMP incorporated, but unlike the normal 4S intermediates of DNA synthesis, these repair products are not precursors to high molecular weight DNA but are further degraded. The high levels of dUTPase as well as the presence of RNA primers on most nascent DNA pieces (Tseng and Goulian, 1977) suggest that repair of uracil-containing DNA does not contribute to the generation of the small, nascent DNA pieces found during DNA synthesis in this in vitro system.     

Dispersive segregation of nucleosomes during replication of simian virus 40 chromosomes

The distribution of preformed ("old") histone octamers between the two arms of DNA replication forks was analyzed in simian virus 40(SV40)-infected cells following treatment with cycloheximide to prevent nucleosome assembly from nascent histones. Viral chromatin synthesized in the presence of cycloheximide was shown to be deficient in nucleosomes. Replicating SV40 DNA (wild-type 800 and capsid assembly mutant, tsB11) was radiolabeled in either intact cells or nuclear extracts supplemented with cytosol. Nascent nucleosomal monomers were then released by extensive digestion of isolated nuclei, nuclear extracts or isolated viral chromosomes with micrococcal nuclease. The labeled nucleosomal DNA was purified and found to hybridize to both strands of SV40 DNA restriction fragments taken from each side of the origin of DNA replication, whereas Okazaki fragments hybridized only to the strand representing the retrograde DNA template. In addition, isolated, replicating SV40 chromosomes were digested with two strand-specific exonucleases that excised nascent DNA from either the forward or the retrograde side of replication forks. Pretreatment of cells with cycloheximide did not result in an excess of prenucleosomal DNA on either side of replication forks, but did increase the amount of internucleosomal DNA. These data are consistent with a dispersive model for nucleosome segregation in which "old" histone octamers are distributed to both arms of DNA replication forks.     

Aberrant DNA repair and DNA replication due to an inherited enzymatic defect in human DNA ligase I.

Two missense mutations in different alleles of the DNA ligase I gene have been described in a patient (46BR) with immunodeficiencies and cellular hypersensitivity to DNA-damaging agents. One of the mutant alleles produces an inactive protein, while the other encodes an enzyme with some residual activity. A subline of identical phenotype that is homozygous (or hemizygous) for the mutant allele encoding this partially active enzyme has facilitated characterization of the enzymatic defect in 46BR. This subline retains only 3 to 5% of normal DNA ligase I activity. The intermediates in the ligation reaction, DNA ligase I-AMP and nicked DNA-AMP, accumulate in vitro and in vivo. The defect of the 46BR enzyme lies primarily in conversion of nicked DNA-AMP into the final ligated DNA product. Assays of DNA repair in 46BR cell extracts and of DNA replication in permeabilized cells have clarified functional roles of DNA ligase I. The initial rate of ligation of Okazaki fragments during DNA replication is apparently normal in 46BR cells, but 25 to 30% of the fragments remain in low-molecular-weight form for prolonged times. DNA base excision repair by 46BR cell extracts shows a delay in ligation and an anomalously long repair patch size that is reduced upon addition of purified normal DNA ligase I.     

Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA. When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands. When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations. DNA synthesis was decreased. In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments. All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract. Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E. coli DNA polymerase I) increased the joining function of the nuclei. The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication.     

Structure of chromatin at deoxyribonucleic acid replication forks: prenucleosomal deoxyribonucleic acid is rapidly excised from replicating simian virus 40 chromosomes by micrococcal nuclease

Replicating simian virus 40 (SV40) chromosomes were found to be similar to other eukaryotic chromosomes in that the rate and extent of micrococcal nuclease (MNase) digestion were greater with replicating than with nonreplicating mature SV40 chromatin. MNase digestion of replicating SV40 chromosomes, pulse labeled in either intact cells or nuclear extracts, resulted in the rapid release of nascent DNA as essentially bare fragments of duplex DNA (3-7S) that had an average length of 120 base pairs and were degraded during the course of the reaction. In addition, nucleosomal monomers, equivalent in size to those from mature chromosomes, were released. On the other hand, MNase digestion of uniformly labeled mature SV40 chromosomes resulted in the release of only nucleosomal monomers and oligomers. The small nascent DNA fragments released from replicating chromosomes represented prenucleosomal DNA (PN-DNA) from the region of replication forks that encompasses the actual sites of DNA synthesis and includes Okazaki fragments. Predigestion of replicating SV40 chromosomes with both Escherichia coli exonuclease III (3'-5') and bacteriophage T7 gene 6 exonuclease (5'-3') resulted in complete degradation of PN-DNA. This result, together with the observation that isolated PN-DNA annealed equally well to both strands of SV40 restriction fragments, demonstrated that PN-DNA originates from both sides of replication forks. Over 90% of isolated Okazaki fragments annealed only to the retrograde DNA template. The characteristics of isolated PN-DNA were assessed by examining its sensitivity to MNase and single strand specific S1 endonuclease, sedimentation behavior before and after deproteinization, buoyant density in CsCl after formaldehyde treatment, and size on agarose gels. In addition, it was observed that MNase digestion of purified SV40 DNA also resulted in the release of a transient intermediate similar in size to PN-DNA, indicating that a DNA-protein complex is not required to account for the appearance of PN-DNA. These and other data provide a model of replicating chromosomes in which DNA synthesis occurs on a region of replication forks that is free of nucleosomes and is designated as prenucleosomal DNA.     

A conserved interaction between the replicative clamp loader and DNA ligase in eukaryotes: implications for Okazaki fragment joining

The recruitment of DNA ligase I to replication foci and the efficient joining of Okazaki fragments is dependent on the interaction between DNA ligase I and proliferating cell nuclear antigen (PCNA). Although the PCNA sliding clamp tethers DNA ligase I to nicked duplex DNA circles, the interaction does not enhance DNA joining. This suggests that other factors may be involved in the joining of Okazaki fragments. In this study, we describe an association between replication factor C (RFC), the clamp loader, and DNA ligase I in human cell extracts. Subsequently, we demonstrate that there is a direct physical interaction between these proteins that involves both the N- and C-terminal domains of DNA ligase I, the N terminus of the large RFC subunit p140, and the p36 and p38 subunits of RFC. Although RFC inhibited DNA joining by DNA ligase I, the addition of PCNA alleviated inhibition by RFC. Notably, the effect of PCNA on ligation was dependent on the PCNA-binding site of DNA ligase I. Together, these results provide a molecular explanation for the key in vivo role of the DNA ligase I/PCNA interaction and suggest that the joining of Okazaki fragments is coordinated by pairwise interactions among RFC, PCNA, and DNA ligase I.     

Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase δ

Lagging strand DNA replication requires the concerted actions of DNA polymerase δ, Fen1 and DNA ligase I for the removal of the RNA/DNA primers before ligation of Okazaki fragments. To better understand this process in human cells, we have reconstituted Okazaki fragment processing by the short flap pathway in vitro with purified human proteins and oligonucleotide substrates. We systematically characterized the key events in Okazaki fragment processing: the strand displacement, Pol δ/Fen1 combined reactions for removal of the RNA/DNA primer, and the complete reaction with DNA ligase I. Two forms of human DNA polymerase δ were studied: Pol δ4 and Pol δ3, which represent the heterotetramer and the heterotrimer lacking the p12 subunit, respectively. Pol δ3 exhibits very limited strand displacement activity in contrast to Pol δ4, and stalls on encounter with a 5′-blocking oligonucleotide. Pol δ4 and Pol δ3 exhibit different characteristics in the Pol δ/Fen1 reactions. While Pol δ3 produces predominantly 1 and 2 nt cleavage products irrespective of Fen1 concentrations, Pol δ4 produces cleavage fragments of 1–10 nts at low Fen1 concentrations. Pol δ3 and Pol δ4 exhibit comparable formation of ligated products in the complete system. While both are capable of Okazaki fragment processing in vitro, Pol δ3 exhibits ideal characteristics for a role in Okazaki fragment processing. Pol δ3 readily idles and in combination with Fen1 produces primarily 1 nt cleavage products, so that nick translation predominates in the removal of the blocking strand, avoiding the production of longer flaps that require additional processing. These studies represent the first analysis of the two forms of human Pol δ in Okazaki fragment processing. The findings provide evidence for the novel concept that Pol δ3 has a role in lagging strand synthesis, and that both forms of Pol δ may participate in DNA replication in higher eukaryotic cells.     

The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation

During replication, Okazaki fragment maturation is a fundamental process that joins discontinuously synthesized DNA fragments into a contiguous lagging strand. Efficient maturation prevents repeat sequence expansions, small duplications, and generation of double-stranded DNA breaks. To address the components required for the process in Thermococcus, Okazaki fragment maturation was reconstituted in vitro using purified proteins from Thermococcus species 9°N or cell extracts. A dual color fluorescence assay was developed to monitor reaction substrates, intermediates, and products. DNA polymerase D (polD) was proposed to function as the replicative polymerase in Thermococcus replicating both the leading and the lagging strands. It is shown here, however, that it stops before the previous Okazaki fragments, failing to rapidly process them. Instead, Family B DNA polymerase (polB) was observed to rapidly fill the gaps left by polD and displaces the downstream Okazaki fragment to create a flap structure. This flap structure was cleaved by flap endonuclease 1 (Fen1) and the resultant nick was ligated by DNA ligase to form a mature lagging strand. The similarities to both bacterial and eukaryotic systems and evolutionary implications of archaeal Okazaki fragment maturation are discussed.     

DNA ligase I and Nbs1 proteins associate in a complex and colocalize at replication factories

DNA ligase I is the main DNA ligase activity involved in eukaryotic DNA replication acting in the joining of Okazaki fragments. This enzyme is also implicated in nucleotide excision repair and in the long-patch base excision repair while its role in the recombinational repair pathways is poorly understood. DNA ligase I is phosphorylated during cell cycle at several serine and threonine residues that regulate its participation in different DNA transactions by modulating the interaction with different protein partners. Here we use an antibody-based array method to identify novel DNA ligase-interacting partners. We show that DNA ligase I participates in several multiprotein complexes with proteins involved in DNA replication and repair, cell cycle control, and protein modification. In particular we demonstrate that DNA ligase I complexes with Nbs1, a core component of the MRN complex critical for detection, processing and repair of double-stranded DNA breaks. The analysis of epitope tagged DNA ligase I mutants demonstrates that the association is mediated by the catalytic fragment of the enzyme. DNA ligase I and Nbs1 colocalize at replication factories during unperturbed replication and after treatment with DNA damaging agents. Since MRN complex is involved in the repair of double-stranded DNA breaks by homologous recombination at stalled replication forks our data support the notion that DNA ligase I participates in homology dependent pathways that deal with replication-associated lesions generated when replication fork encounters DNA damage.     

Mechanism of DNA chain growth: XV. RNA-linked nascent DNA pieces in Escherichia coli strains assayed with spleen exonuclease

A new method for the detection and assay of RNA-linked nascent DNA pieces has been developed. The method relies on selective degradation by spleen exonuclease of radioactive 5′-OH terminated DNA produced from the pulse-labelled nascent pieces upon alkaline hydrolysis. Analysis with this method in wild type Escherichia coli has shown relatively high proportions of the RNA-linked molecules after shorter pulses and in the smaller pieces, supporting the transient nature of the RNA attachment to the nascent pieces. The RNA-linked nascent DNA pieces are accumulated by both E. coli polAex1 (defective in 5′ → 3′ exonuclease of DNA polymerase I) and E. coli polA12 and polA1 (defective in polymerase of DNA polymerase I), suggesting the requirement of the concerted action of both 5′ → 3′ exonuclease and polymerase of DNA polymerase I for the removal of the RNA attached to the nascent pieces. Most of the nascent DNA pieces accumulated by E. coli ligts7 (defective in DNA ligase) are not linked to RNA, as expected from the direct role of DNA ligase in joining of the pieces. The analysis also has shown that a large portion of the nascent DNA pieces present in the cell under the normal steady-state conditions are not linked to RNA and that the level of the RNA-free DNA pieces is also increased in polA mutants. These findings suggest that the removal of RNA from the nascent pieces is a relatively rapid process and the joining reaction is a rate-limiting step that requires the concurrent action of DNA polymerase and DNA ligase.     

Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40

Agents discriminating between DNA polymerase alpha and DNA polymerases of class delta (polymerase delta or epsilon) were used to characterize steps in the synthesis of the lagging DNA strand of simian virus 40 during DNA replication in isolated nuclei. The synthesis of lagging-strand intermediates below 40 nucleotides, termed DNA primers (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988), was selectively inhibited by butylphenyl dGTP or by neutralizing DNA polymerase alpha monoclonal antibodies. The synthesis of longer lagging chains of up to 250 nucleotides (Okazaki pieces) was affected to a lesser extent, possibly indirectly, by these agents. Aphidicolin, which inhibits both alpha- and delta-class enzymes, elicited the opposite pattern: DNA primers accumulated in its presence and were not converted into Okazaki pieces. These and previous data suggest that DNA polymerase alpha primase synthesizes DNA primers, whereas another DNA polymerase, presumably DNA polymerase delta or epsilon, mediates the conversion of DNA primers into Okazaki pieces.