Lagging Strand Synthesis Research Articles

Replication proteins influence the maintenance of telomere length and telomerase protein stability.

We investigated the effects of fission yeast replication genes on telomere length maintenance and identified 20 mutant alleles that confer lengthening or shortening of telomeres. The telomere elongation was telomerase dependent in the replication mutants analyzed. Furthermore, the telomerase catalytic subunit, Trt1, and the principal initiation and lagging-strand synthesis DNA polymerase, Polalpha, were reciprocally coimmunoprecipitated, indicating these proteins physically coexist as a complex in vivo. In a polalpha mutant that exhibited abnormal telomere lengthening and slightly reduced telomere position effect, the cellular level of the Trt1 protein was significantly lower and the coimmunoprecipitation of Trt1 and Polalpha was severely compromised compared to those in the wild-type polalpha cells. Interestingly, ectopic expression of wild-type polalpha in this polalpha mutant restored the cellular Trt1 protein to the wild-type level and shortened the telomeres to near-wild-type length. These results suggest that there is a close physical relationship between the replication and telomerase complexes. Thus, mutation of a component of the replication complex can affect the telomeric complex in maintaining both telomere length equilibrium and telomerase protein stability.

Preparation and crystallization of human flap endonuclease FEN-1 in complex with proliferating-cell nuclear antigen, PCNA.

Flap endonuclease-1 (FEN-1) is a structure-specific nuclease that removes 5'-overhanging flaps in DNA repair and replication. FEN-1 binds proliferating-cell nuclear antigen (PCNA), a DNA-clamp protein, when processing Okazaki fragments during lagging-strand DNA synthesis. Here, the crystallization of the complex between human FEN-1 and PCNA is reported. The crystals were found to belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 82.2, b = 143.4, c = 246.7 A, and contained one complex in the crystallographic asymmetric unit. A diffraction data set was collected to a resolution of 3.0 A.

Budding yeastmcm10/dna43mutant requires a novel repair pathway for viability

MCM10 is essential for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae. Mcm10p functionally interacts with components of the pre-replicative complex (Mcm2-Mcm7 complex and origin recognition complex) as well as the pre-initiation complex component (Cdc45p) suggesting that it may be a component of the pre-RC as well as the pre-IC. Two-dimensional gel electrophoresis analysis showed that Mcm10p is required not only for the initiation of DNA synthesis at replication origins but also for the smooth passage of replication forks at origins. Genetic analysis showed that MCM10 interacts with components of the elongation machinery such as Pol delta and Pol epsilon, suggesting that it may play a role in elongation replication. We show that the mcm10 mutation causes replication fork pausing not only at potentially active origins but also at silent origins. We screened for mutations that are lethal in combination with mcm10-1 and obtained seven mutants named slm1-slm6 for synthetically lethal with mcm10. These mutants comprised six complementation groups that can be divided into three classes. Class 1 includes genes that encode components of the pre-RC and pre-IC and are represented by SLM3, 4 and 5 which are allelic to MCM7, MCM2 and CDC45, respectively. Class 2 includes genes involved in the processing of Okazaki fragments in lagging strand synthesis and is represented by SLM1, which is allelic to DNA2. Class 3 includes novel DNA repair genes represented by SLM2 and SLM6. The viability of the mcm10-1 mutant is dependent on a novel repair pathway that may participate either in resolving accumulated replication intermediates or the damage caused by blocked replication forks. These results are consistent with the hypothesis that Mcm10p is required for the passage of replication forks through obstacles such as those created by pre-RCs assembled at active or inactive replication origins.

Fate of DNA replication fork encountering a single DNA lesion during oriC plasmid DNA replication in vitro.

The inhibition of DNA replication fork progression by DNA lesions can lead to cell death or genome instability. However, little is known about how such DNA lesions affect the concurrent synthesis of leading- and lagging-strand DNA catalysed by the protein machinery used in chromosomal replication. Using a system of semi-bidirectional DNA replication of an oriC plasmid that employs purified replicative enzymes and a replication-terminating protein of Escherichia coli, we examined the dynamics of the replication fork when it encounters a single abasic DNA lesion on the template DNA. A DNA lesion located on the lagging strand completely blocked the synthesis of the Okazaki fragment extending toward the lesion site, but did not affect the progression of the replication fork or leading-strand DNA synthesis. In contrast, a DNA lesion on the leading strand stalled the replication fork in conjunction with strongly inhibiting leading-strand synthesis. However, about two-thirds of the replication forks encountering this lesion maintained lagging-strand synthesis for about 1 kb beyond the lesion site, and the velocity with which the replication fork progressed seemed to be significantly reduced. The blocking DNA lesion affects DNA replication differently depending on which strand, leading or lagging, contains the lesion.

Mitochondrial DNA replication: what we know.

Since the isolation and physical characterization of mammalian mitochondrial DNA (mtDNA) over 35 years ago, numerous works have been published that have examined its physical structure and properties, including its mode of replication and transcription. The established replication model posits that leading-strand replication of mammalian mtDNA begins at closely spaced, defined sites located downstream from a major transcription promoter and proceeds unidirectionally with displacement of the parental leading strand until approximately two-thirds of the closed circular mtDNA has been copied. As a consequence, the replication fork passes a major origin for lagging-strand synthesis, exposing it in single-stranded form. Displacement as a single-strand is thought to allow the characteristic secondary structure of this origin to occur, thereby permitting initiation of lagging-strand synthesis. A natural consequence of the separate and distinct locations of the two origins is that the two segregated progeny mtDNA circles are of two types: one a duplex circle with a newly synthesized leading strand and the other a gapped circle with a partial newly synthesized lagging strand. In each case, the final steps of synthesis and ligation result in closed circular mtDNA products. Recently, mammalian mtDNA isolates have been subjected to 2D-gel electrophoretic analysis in attempts to assign features to mtDNA molecules that, by virtue of their anomalous migration behavior, could infer them to be candidates for replication intermediates. This review will describe the essential features of the historical findings of mammalian mtDNA replication studies and integrate the more recent observations in developing a current model for this process.

Mechanism of the E. coli τ Processivity Switch during Lagging-Strand Synthesis

The E. coli replication machinery employs a β clamp that tethers the polymerase to DNA, thus ensuring high processivity. The replicase also contains a processivity switch that dissociates the polymerase from its β clamp. The switch requires the τ subunit of the clamp loader and is regulated by different DNA structures. At a primed site, the switch is “off.” When the replicase reaches the downstream primer to form a nick, the switch is flipped, and τ ejects the polymerase from β. This switch has high fidelity for completed synthesis, remaining “off” until just prior to incorporation of the last nucleotide and turning “on” only after addition of the last dNTP. These actions of τ are confined to its C-terminal region, which is located outside the clamp loading apparatus. Thus, this highly processive replication machine has evolved a mechanism to specifically counteract processivity at a defined time in the lagging-strand cycle.

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin.

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.

Protein-Protein Interactions in the Bacteriophage T4 Replisome: THE LEADING STRAND HOLOENZYME IS PHYSICALLY LINKED TO THE LAGGING STRAND HOLOENZYME AND THE PRIMOSOME

The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.

Differential processing of leading- and lagging-strand ends at Saccharomyces cerevisiae telomeres revealed by the absence of Rad27p nuclease.

Saccharomyces cerevisiae strains lacking the Rad27p nuclease, a homolog of the mammalian FEN-1 protein, display an accumulation of extensive single-stranded G-tails at telomeres. Furthermore, the lengths of telomeric repeats become very heterogeneous. These phenotypes could be the result of aberrant Okazaki fragment processing of the C-rich strand, elongation of the G-rich strand by telomerase, or an abnormally high activity of the nucleolytic activities required to process leading-strand ends. To distinguish among these possibilities, we analyzed strains carrying a deletion of the RAD27 gene and also lacking genes required for in vivo telomerase activity. The results show that double-mutant strains died more rapidly than strains lacking only telomerase components. Furthermore, in such strains there is a significant reduction in the signals for G-tails as compared to those detected in rad27delta cells. The results from studies of the replication intermediates of a linear plasmid in rad27delta cells are consistent with the idea that only one end of the plasmid acquires extensive G-tails, presumably the end made by lagging-strand synthesis. These data further support the notion that chromosome ends have differential requirements for end processing, depending on whether the ends were replicated by leading- or lagging-strand synthesis.

On the Specificity of Interaction between the Saccharomyces cerevisiae Clamp Loader Replication Factor C and Primed DNA Templates during DNA Replication

Replication factor C (RFC) catalyzes assembly of circular proliferating cell nuclear antigen clamps around primed DNA, enabling processive synthesis by DNA polymerase during DNA replication and repair. In order to perform this function efficiently, RFC must rapidly recognize primed DNA as the substrate for clamp assembly, particularly during lagging strand synthesis. Earlier reports as well as quantitative DNA binding experiments from this study indicate, however, that RFC interacts with primer-template as well as single- and double-stranded DNA (ssDNA and dsDNA, respectively) with similar high affinity (apparent K(d) approximately 10 nm). How then can RFC distinguish primed DNA sites from excess ssDNA and dsDNA at the replication fork? Further analysis reveals that despite its high affinity for various DNA structures, RFC selects primer-template DNA even in the presence of a 50-fold excess of ssDNA and dsDNA. The interaction between ssDNA or dsDNA and RFC is far less stable than between primed DNA and RFC (k(off) > 0.2 s(-1) versus 0.025 s(-1), respectively). We propose that the ability to rapidly bind and release single- and double-stranded DNA coupled with selective, stable binding to primer-template DNA allows RFC to scan DNA efficiently for primed sites where it can pause to initiate clamp assembly.

Retrotransposition of the Ll.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets.

Catalytic group II introns are mobile retroelements that invade cognate intronless genes via retrohoming, where the introns reverse splice into double-stranded DNA (dsDNA) targets. They can also retrotranspose to ectopic sites at low frequencies. Whereas our previous studies with a bacterial intron, Ll.LtrB, supported frequent use of RNA targets during retrotransposition, recent experiments with a retrotransposition indicator gene indicate that DNA, rather than RNA, is a prominent target, with both dsDNA and single-stranded DNA (ssDNA) as possibilities. Thus retrotransposition occurs in both transcriptional sense and antisense orientations of target genes, and is largely independent of homologous DNA recombination and of the endonuclease function of the intron-encoded protein, LtrA. Models based on both dsDNA and ssDNA targeting are presented. Interestingly, retrotransposition is biased toward the template for lagging-strand DNA synthesis, which suggests the possibility of the replication folk as a source of ssDNA. Consistent with some use of ssDNA targets, many retrotransposition sites lack nucleotides critical for the unwinding of target duplex DNA. Moreover, in vitro the intron reverse spliced into ssDNA more efficiently than dsDNA substrates for some of the retrotransposition sites. Furthermore, many bacterial group II introns reside on the lagging-strand template, hinting at a role for DNA replication in intron dispersal in nature.

Asymmetry of DNA replication and translesion synthesis of UV-induced thymine dimers

In vitro replication assays for detection and quantification of bypass of UV-induced DNA photoproducts were used to compare the capacity of extracts prepared from different human cell lines to replicate past the cis,syn cyclobutane thymine dimer ([ c,s]TT). The results demonstrated that neither nucleotide excision repair (NER) nor mismatch repair (MMR) activities in the intact cells interfered with measurements of bypass replication efficiencies in vitro. Extracts prepared from HeLa (NER- and MMR-proficient), xeroderma pigmentosum group A (NER-deficient), and HCT116 (MMR-deficient) cells displayed similar capacity for translesion synthesis, when the substrate carried the site-specific [ c,s]TT on the template for the leading or the lagging strand of nascent DNA. Extracts from xeroderma pigmentosum variant cells, which lack DNA polymerase η, were devoid of bypass activity. Bypass-proficient extracts as a group ( n=16 for 3 extracts) displayed higher efficiency ( P=0.005) for replication past the [ c,s]TT during leading strand synthesis (84±22%) than during lagging strand synthesis (64±13%). These findings are compared to previous results concerning the bypass of the (6–4) photoproduct [Biochemistry 40 (2001) 15215] and analyzed in the context of the reported characteristics of bypass DNA polymerases implicated in translesion synthesis of UV-induced DNA lesions. Models to explain how these enzymes might interact with the DNA replication machinery are considered. An alternative pathway of bypass replication, which avoids translesion synthesis, and the mutagenic potential of post-replication repair mechanisms that contribute to the duplication of the human genome damaged by UV are discussed.

Characterization of DNA synthesis catalyzed by bacteriophage T4 replication complexes reconstituted on synthetic circular substrates.

Replication complexes were reconstituted using the eight purified bacteriophage T4 replication proteins and synthetic circular 70-, 120- or 240-nt DNA substrates annealed to a leading-strand primer. To differentiate leading strands from lagging strands, the circular parts of the substrates lacked dCMP; thus, no dCTP was required for leading-strand synthesis and no dGTP for lagging-strand synthesis. The size of the substrates was crucial, the longer substrates supporting much more DNA synthesis. Leading and lagging strands were synthesized in a coupled manner. Specifically targeting leading-strand synthesis by decreasing the concentration of dGTP decreased the rate of extension of leading strands. However, blocking lagging-strand synthesis by lowering the dCTP concentration, by omitting dCTP altogether, by adding ddCTP, or with a single abasic site had no immediate effect on the rate of extension of leading strands.

Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects.

Checkpoint-mediated control of replicating chromosomes is essential for preventing cancer. In yeast, Rad53 kinase protects stalled replication forks from pathological rearrangements. To characterize the mechanisms controlling fork integrity, we analyzed replication intermediates formed in response to replication blocks using electron microscopy. At the forks, wild-type cells accumulate short single-stranded regions, which likely causes checkpoint activation, whereas rad53 mutants exhibit extensive single-stranded gaps and hemi-replicated intermediates, consistent with a lagging-strand synthesis defect. Further, rad53 cells accumulate Holliday junctions through fork reversal. We speculate that, in checkpoint mutants, abnormal replication intermediates begin to form because of uncoordinated replication and are further processed by unscheduled recombination pathways, causing genome instability.

Disruption of dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA.

Genetic integrity is crucial to normal cell function, and mutations in genes required for DNA replication and repair underlie various forms of genetic instability and disease, including cancer. One structural feature of intact genomes is runs of homopolymeric dC/dG. Here we describe an unusual mutator phenotype in Caenorhabditis elegans characterized by deletions that start around the 3' end of polyguanine tracts and terminate at variable positions 5' from such tracts. We observed deletions throughout genomic DNA in about half of polyguanine tracts examined, especially those containing 22 or more consecutive guanine nucleotides. The mutator phenotype results from disruption of the predicted gene F33H2.1, which encodes a protein with characteristics of a DEAH helicase and which we have named dog-1 (for deletions of guanine-rich DNA). Nematodes mutated in dog-1 showed germline as well as somatic deletions in genes containing polyguanine tracts, such as vab-1. We propose that DOG-1 is required to resolve the secondary structures of guanine-rich DNA that occasionally form during lagging-strand DNA synthesis.

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage T7 replication proteins

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.

Mitochondrial DNA structure and function

This chapter discusses mitochondrial DNA (mtDNA) structures and its functions. The human mtDNA is representative of mammalian mitochondrial genomes. Comparison of the nucleotide sequences of mammal mtDNA revealed some degree of conservation in the promoter regions as well as in three other regions (termed “Conserved Sequence Blocks,” or CSB I, CSB II, and CSB III). The mammalian mitochondrial genome has two modes of replication. The first one involves the asymmetric replication of the leading and lagging strands. The second one, based on the observation of replication intermediates in two-dimensional (2D) gels suggested that replication, in a certain number of mtDNA molecules, involves coupled leading- and lagging-strand synthesis. Many factors involved in mammalian mtDNA replication have been characterized. Because of its probable prokaryote origin, in many aspects, the mitochondrion behaves as an independent entity living inside an eukaryotic cell. All basic processes associated with life (DNA maintenance, transcription, and translation) occur inside the organelle. However, the vast majority of the factors involved in promoting and controlling these processes are borrowed from the cytoplasm, where nuclear-coded proteins are synthesized.

ATP-dependent DNA ligases.

By catalyzing the joining of breaks in the phosphodiester backbone of duplex DNA, DNA ligases play a vital role in the diverse processes of DNA replication, recombination and repair. Three related classes of ATP-dependent DNA ligase are readily apparent in eukaryotic cells. Enzymes of each class comprise catalytic and non-catalytic domains together with additional domains of varying function. DNA ligase I is required for the ligation of Okazaki fragments during lagging-strand DNA synthesis, as well as for several DNA-repair pathways; these functions are mediated, at least in part, by interactions between DNA ligase I and the sliding-clamp protein PCNA. DNA ligase III, which is unique to vertebrates, functions both in the nucleus and in mitochondria. Two distinct isoforms of this enzyme, differing in their carboxy-terminal sequences, are produced by alternative splicing: DNA ligase IIIalpha has a carboxy-terminal BRCT domain that interacts with the mammalian DNA-repair factor XrccI, but both alpha and beta isoforms have an amino-terminal zinc-finger motif that appears to play a role in the recognition of DNA secondary structures that resemble intermediates in DNA metabolism. DNA ligase IV is required for DNA non-homologous end joining pathways, including recombination of the V(D)J immunoglobulin gene segments in cells of the mammalian immune system. DNA ligase IV forms a tight complex with Xrcc4 through an interaction motif located between a pair of carboxy-terminal BRCT domains in the ligase. Recent structural studies have shed light on the catalytic function of DNA ligases, as well as illuminating protein-protein interactions involving DNA ligases IIIalpha and IV.

Bypass replication in vitro of UV-induced photoproducts blocking leading or lagging strand synthesis.

In vitro replication assays were used to determine the capacity of HeLa extracts to replicate past one of the two major photoproducts produced by ultraviolet radiation at adjacent thymines in duplex DNA, namely, the cis,syn cyclobutane dimer ([c,s]TT) and the 6-4 pyrimidine-pyrimidone adduct ([6-4]TT). The site-specific photoproduct was placed on the template either to the leading strand or to the lagging strand of nascent DNA with respect to the first fork encountering the lesion during bidirectional replication of closed circular duplex molecules carrying the SV40 origin. Replication products from time-course reactions were fractionated by gel electrophoresis in the presence of ethidium bromide. Recognition and quantification of true translesion synthesis products, i.e., newly synthesized closed circular molecules carrying the photoproduct, were aided by specific substrate modifications (a T:T mismatch in a unique PstI site nearby the photoproduct) and improved assay conditions (internal standard to control for completion of PstI digestion). Extracts from HeLa cells, which express DNA polymerase eta, were competent to replicate past the [c,s]TT on either strand. The efficiency of bypass replication of the [c,s]TT on the template to the leading or the lagging strand was 71% and 67%, respectively. The same extracts demonstrated very low efficiency of translesion synthesis (at most 8-10%) of the [6-4]TT on either template position. Replication-competent cell-free extracts from other human cells were also deficient in the bypass of the [6-4]TT in vitro.

In vitro reconstitution of the end replication problem.

The end replication problem hypothesis proposes that the ends of linear DNA cannot be replicated completely during lagging strand DNA synthesis. Although the idea has been widely accepted for explaining telomere attrition during cell proliferation, it has never been directly demonstrated. In order to take a biochemical approach to understand how linear DNA ends are replicated, we have established a novel in vitro linear simian virus 40 DNA replication system. In this system, terminally biotin-labeled linear DNAs are conjugated to avidin-coated beads and subjected to replication reactions. Linear DNA was efficiently replicated under optimized conditions, and replication products that had replicated using the original DNA templates were specifically analyzed by purifying bead-bound replication products. By exploiting this system, we showed that while the leading strand is completely synthesized to the end, lagging strand synthesis is gradually halted in the terminal approximately 500-bp region, leaving 3' overhangs. This result is consistent with observations in telomerase-negative mammalian cells and formally demonstrates the end replication problem. This study provides a basis for studying the details of telomere replication.