Replication of mitochondrial DNA occurs throughout the mitochondria of cultured human cells

Replication of mitochondrial DNA (mtDNA) is dependent on nuclear-encoded factors. It has been proposed that this reliance may exert spatial restrictions on the sites of mtDNA replication within the cytoplasm, as a previous study only detected mtDNA synthesis in perinuclear mitochondria. We have studied mtDNA replication in situ in a variety of human cell cultures labeled with 5-bromo-2'-deoxyuridine. In contrast to what has been reported, mtDNA synthesis was detected at multiple sites throughout the mitochondrial network following short pulses with bromodeoxyuridine. Although no bromodeoxyuridine incorporation was observed in anuclear platelets, incorporation into mtDNA of fibroblasts that had been enucleated 2 h prior to labeling was readily detectable. Blotting experiments indicated that the bromodeoxyuridine incorporation into mtDNA observed in situ represents replication of the entire mtDNA molecule. The studies also showed that replication of mtDNA occurred at any stage of the cell cycle in proliferating cells and continued in postmitotic cells, although at a lower level. These results demonstrate that mtDNA replication is not restricted to mitochondria in the proximity of the nucleus and imply that all components of the replication machinery are available at sufficient levels throughout the mitochondrial network to permit mtDNA replication throughout the cytoplasm.     

Replication of the lagging strand: a concert of at least 23 polypeptides.

DNA replication is one of the most important events in living cells, and it is still a key problem how the DNA replication machinery works in its details. A replication fork has to be a very dynamic apparatus since frequent DNA polymerase switches from the initiating DNA polymerase alpha to the processive elongating DNA polymerase delta occur at the leading strand (about 8 x 10(4) fold on both strands in one replication round) as well as at the lagging strand (about 2 x 10(7) fold on both strands in one replication round) in mammalian cells. Lagging strand replication involves a very complex set of interacting proteins that are able to frequently initiate, elongate and process Okazaki fragments of 180 bp. Moreover, key proteins of this important process appear to be controlled by S-phase check-point proteins. It became furthermore clear in the last few years that DNA replication cannot be considered uncoupled from DNA repair, another very important event for any living organism. The reconstitution of nucleotide excision repair and base excision repair in vitro with purified components clearly showed that the DNA synthesis machinery of both of these macromolecular events are similar and do share many components of the lagging strand DNA synthesis machinery. In this minireview we summarize our current knowledge of the components involved in the execution and regulation of DNA replication at the lagging strand of the replication fork.     

Repetitive lagging strand DNA synthesis by the bacteriophage T4 replisome

Our studies on the T4 replisome build on the seminal work from the Alberts laboratory. They discovered essentially all the proteins that constitute the T4 replisome, isolated them, and measured their enzymatic activities. Ultimately, in brilliant experiments they reconstituted in vitro a functioning replisome and in the absence of structural information created a mosaic as to how such a machine might be assembled. Their consideration of the problem of continuous leading strand synthesis opposing discontinuous lagging strand synthesis led to their imaginative proposal of the trombone model, an illustration that graces all textbooks of biochemistry. Our subsequent work deepens their findings through experiments that focus on defining the kinetics, structural elements, and protein-protein contacts essential for replisome assembly and function. In this highlight we address when Okazaki primer synthesis is initiated and how the primer is captured by a recycling lagging strand polymerase--problems that the Alberts laboratory likewise found mysterious and significant for all replisomes.     

Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase

The eukaryotic replicative DNA helicase, CMG, unwinds DNA by an unknown mechanism. In some models, CMG encircles and translocates along one strand of DNA while excluding the other strand. In others, CMG encircles and translocates along duplex DNA. To distinguish between these models, replisomes were confronted with strand-specific DNA roadblocks in Xenopus egg extracts. An ssDNA translocase should stall at an obstruction on the translocation strand but not the excluded strand, whereas a dsDNA translocase should stall at obstructions on either strand. We found that replisomes bypass large roadblocks on the lagging strand template much more readily than on the leading strand template. Our results indicate that CMG is a 3' to 5' ssDNA translocase, consistent with unwinding via "steric exclusion." Given that MCM2-7 encircles dsDNA in G1, the data imply that formation of CMG in S phase involves remodeling of MCM2-7 from a dsDNA to a ssDNA binding mode.     

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.     

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle‐dependent recruitment of telomere‐specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S‐phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase ε (Polε) arrived at telomeres earlier than the lagging strand DNA polymerases α (Polα) and δ (Polδ). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polε, whereas S‐phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polα. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.     

Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.     

Replication strand preference for deletions associated with DNA palindromes

We have isolated and sequenced a set of deletions stimulated by DNA palindromes in Escherichia coli . All of the deletions are asymmetric with respect to the parental sequence and have occurred at short direct repeats. This is consistent with deletion by strand slippage during DNA replication. The orientation of the asymmetry in such deletion products is diagnostic of the direction of the strand slippage event. It is therefore also diagnostic of its occurrence on the leading or lagging strand of the replication fork when the direction of replication is known. In all cases in which the orientation of the asymmetry could be determined with respect to DNA replication, the products were consistent with a preference for deletion on the lagging strand of the fork. The data include replication slippage in three situations: on the chromosome of E . coli , in bacteriophage λ and in high-copy-number pUC-based plasmids.     

Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.     

Eukaryotic lagging strand DNA replication employs a multi-pathway mechanism that protects genome integrity

In eukaryotic nuclear DNA replication, one strand of DNA is synthesized continuously, but the other is made as Okazaki fragments that are later joined. Discontinuous synthesis is inherently more complex, and fragmented intermediates create risks for disruptions of genome integrity. Genetic analyses and biochemical reconstitutions indicate that several parallel pathways evolved to ensure that the fragments are made and joined with integrity. An RNA primer is removed from each fragment before joining by a process involving polymerase-dependent displacement into a single-stranded flap. Evidence in vitro suggests that, with most fragments, short flaps are displaced and efficiently cleaved. Some flaps can become long, but these are also removed to allow joining. Rarely, a flap can form structure, necessitating displacement of the entire fragment. There is now evidence that post-translational protein modification regulates the flow through the pathways to favor protection of genomic information in regions of actively transcribed chromatin.     

Replication fidelity in E. coli: Differential leading and lagging strand effects for dnaE antimutator alleles

DNA Pol III holoenzyme (HE) is the major DNA replicase of Escherichia coli. It is a highly accurate enzyme responsible for simultaneously replicating the leading- and lagging DNA strands. Interestingly, the fidelity of replication for the two DNA strands is unequal, with a higher accuracy for lagging-strand replication. We have previously proposed this higher lagging-strand fidelity results from the more dissociative character of the lagging-strand polymerase. In support of this hypothesis, an E. coli mutant carrying a catalytic DNA polymerase subunit (DnaE915) characterized by decreased processivity yielded an antimutator phenotype (higher fidelity). The present work was undertaken to gain deeper insight into the factors that influence the fidelity of chromosomal DNA replication in E. coli. We used three different dnaE alleles (dnaE915, dnaE911, and dnaE941) that had previously been isolated as antimutators. We confirmed that each of the three dnaE alleles produced significant antimutator effects, but in addition showed that these antimutator effects proved largest for the normally less accurate leading strand. Additionally, in the presence of error-prone DNA polymerases, each of the three dnaE antimutator strains turned into mutators. The combined observations are fully supportive of our model in which the dissociative character of the DNA polymerase is an important determinant of in vivo replication fidelity. In this model, increased dissociation from terminal mismatches (i.e., potential mutations) leads to removal of the mismatches (antimutator effect), but in the presence of error-prone (or translesion) DNA polymerases the abandoned terminal mismatches become targets for error-prone extension (mutator effect). We also propose that these dnaE alleles are promising tools for studying polymerase exchanges at the replication fork.     

Replication origin of mitochondrial DNA in insects

The precise position of the replication origin (O(R)) of mtDNA was determined for insect species belonging to four different orders (four species of Drosophila, Bombyx mori, Triborium castaneum, and Locusta migratoria, which belong to Diptera, Lepidoptera, Coleoptera, and Orthoptera, respectively). Since the free 5' ends of the DNA strands of mtDNA are interpreted as the O(R), their positions were mapped at 1-nucleotide resolution within the A + T-rich region by using the ligation-mediated PCR method. In all species examined, the free 5' ends were found within a very narrow range of several nucleotides in the A + T-rich region. For four species of Drosophila, B. mori, and T. castaneum, which belong to holometabolous insects, although the O(R)'s were located at different positions, they were located immediately downstream of a series of thymine nucleotides, the so-called T-stretch. These results strongly indicate that the T-stretch is involved in the recognition of the O(R) of mtDNA at least among holometabolous insects. For L. migratoria (hemimetabolous insect), on the other hand, none of the long stretches of T's was found in the upstream portion of the O(R), suggesting that the regulatory sequences involved in the replication initiation process have changed through insect evolution.     

The control mechanism for lagging strand polymerase recycling during bacteriophage T4 DNA replication

Given the polarity of DNA duplex, replication by the leading strand polymerase is continuous whereas that by the lagging strand polymerase is discontinuous proceeding through Okazaki fragments. Yet the respective polymerases act processively, implying that the recycling of the lagging strand polymerase is a controlled process. We demonstrate that the rate of the lagging strand polymerase relative to that of fork movement affects Okazaki fragment size and generates ssDNA gaps. We show by using a substrate with limited priming sites that Okazaki fragments can be shifted to shorter lengths by varying the rate of the primase. We find that clamp and clamp loader levels affect both primer utilization and Okazaki fragment size, possibly implicating clamp loading onto the RNA primer in the mechanism of lagging strand polymerase recycling. We formulate a signaling model capable of rationalizing the distribution of Okazaki fragments under various conditions for this and possibly other replisomes.     

Exposure to camptothecin breaks leading and lagging strand simian virus 40 DNA replication forks

To better understand aberrant simian virus 40 DNA replication intermediates produced by exposure of infected cells to the anticancer drug camptothecin, we compared them to forms produced by S1 nuclease digestion of normal viral replication intermediates. All of the major forms were identical in both cases. Thus the aberrant viral replicating forms in camptothecin-treated cells result from DNA strand breaks at replication forks. Linear simian virus 40 forms which are produced by camptothecin exposure during viral replication were identified as detached DNA replication bubbles. This indicates that double strand DNA breaks caused by camptothecin-topoisomerase I complexes occur at both leading and lagging strand replication forks in vivo.     

Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.