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PubMed2026

Single-molecule tracking of RNA-DNA hybrid removal enzymes important for lagging-strand replication.

The formation of RNA-DNA hybrid (RDH) primers by primase is an essential step in the recruitment of DNA polymerase during replication initiation and for the synthesis of each Okazaki fragment on the lagging strand. In addition to primers, RDHs form through misincorporation of ribonucleotides by DNA polymerase during elongation and by formation of R loops during transcription. R loops are three-stranded structures that form when the nascent mRNA anneals to the template DNA strand, displacing the complementary DNA strand. The persistence of RDHs is deleterious to genome stability in all cells because they increase susceptibility to mutations, impaired replication fork progression, DNA double-stranded breaks, and genomic rearrangements. In many bacteria, it is well established that components of the replicative DNA polymerase form a macromolecular complex that can be imaged using single-molecule or ensemble fluorescence approaches. The spatiotemporal regulation of proteins involved in RDH removal during lagging-strand maturation is less clear. Here, we study three proteins that are involved in the removal of RDHs from the lagging strand during DNA replication in the Gram-positive bacterium Bacillus subtilis: DNA polymerase I (Pol I), FenA, and RNase HIII. We characterized the behavior of each PAmCherry-tagged lagging-strand enzyme in living cells using single-particle tracking photoactivated localization microscopy. We find that all three proteins are highly mobile, suggesting residence times at their target substrates are below our temporal resolution. We also find evidence that Pol I activity is modulated through interaction with the replisome, whereas FenA and RNase HIII are regulated through access to the nucleoid. Our results provide new insight into how enzymes are recruited to resolve RDHs during lagging-strand replication in vivo.

DNA Replication↗

PubMed2005

Gross chromosomal rearrangements and elevated recombination at an inducible site-specific replication fork barrier.

Genomic rearrangements linked to aberrant recombination are associated with cancer and human genetic diseases. Such recombination has indirectly been linked to replication fork stalling. Using fission yeast, we have developed a genetic system to block replication forks at nonhistone/DNA complexes located at a specific euchromatic site. We demonstrate that stalled replication forks lead to elevated intrachromosomal and ectopic recombination promoting site-specific gross chromosomal rearrangements. We show that recombination is required to promote cell viability when forks are stalled, that recombination proteins associate with sites of fork stalling, and that recombination participates in deleterious site-specific chromosomal rearrangements. Thus, recombination is a "double-edged sword," preventing cell death when the replisome disassembles at the expense of genetic stability.

Chromosomes↗

PubMed2005

Stimulation of mutagenesis by proportional deoxyribonucleoside triphosphate accumulation in Escherichia coli.

Intracellular pool sizes of deoxyribonucleoside triphosphates (dNTPs) are highly regulated. Unbalanced dNTP pools, created by abnormal accumulation or deficiency of one nucleotide, are known to be mutagenic and to have other genotoxic consequences. Recent studies in our laboratory on DNA replication in vitro suggested that balanced accumulation of dNTPs, in which all four pools increase proportionately, also stimulates mutagenesis. In this paper, we ask whether proportional dNTP pool increases are mutagenic also in living cells. Escherichia coli was transformed with recombinant plasmids that overexpress E. coli genes nrdA and nrdB, which encode the two protein subunits of aerobic ribonucleotide reductase. Roughly proportional dNTP pool expansion, by factors of 2- to 6-fold in different experiments, was accompanied by increases in spontaneous mutation frequency of up to 40-fold. Expression of a catalytically inactive ribonucleotide reductase had no effect on either dNTP pools or mutagenesis, suggesting that accumulation of dNTPs is responsible for the increased mutagenesis. Preliminary experiments with strains defective in SOS regulon induction suggest a requirement for one or more SOS functions in the dNTP-enhanced mutagenesis. Because a replisome extending from correctly matched 3'-terminal nucleotides is almost certainly saturated with dNTP substrates in vivo, whereas chain extension from mismatched nucleotides almost certainly proceeds at sub-saturating rates, we propose that the mutagenic effect of proportional dNTP pool expansion is preferential stimulation of chain extension from mismatches as a result of increases in intracellular dNTP concentrations.

Bacterial Proteins↗

PubMed2026

Mechanistic diversity of clamp loading at small DNA gaps.

DNA sliding clamps, including PCNA (proliferating cell nuclear antigen) and the 9-1-1 (RAD9-RAD1-HUS1 in humans) complex, are ring-shaped protein complexes that encircle DNA and serve as central interaction platforms in DNA replication, repair, and checkpoint signaling. While clamp loading at canonical primer-template junctions by AAA+ (ATPases associated with diverse cellular activities) clamp loaders is well established, how clamps are loaded onto physiologically relevant but geometrically constrained DNA intermediates, such as nicks and single-stranded gaps, has remained unclear. Recent cryo-electron microscopy studies reveal that clamp loaders have evolved distinct strategies to overcome these constraints and to specialize for different genomic contexts. At gapped DNA, the eukaryotic clamp loader RFC (replication factor C) engages both 3'- and 5'-recessed DNA ends and can locally unwind DNA, enabling PCNA loading across a wide range of gap sizes. In contrast, the bacterial DnaX clamp loader lacks a 5'-DNA-binding site and does not unwind DNA, instead loading the β-clamp at small gaps (<6 nt) by sharply bending DNA. The checkpoint clamp loader Rad24-RFC (RAD17-RFC in humans) similarly lacks DNA unwinding activity, restricting loading of 9-1-1 clamp to larger gaps (≥6 nt). In a distinct specialization, Ctf18-RFC interacts with the leading-strand DNA polymerase ε, positioning it as a dedicated loader for leading-strand synthesis, whereas Elg1-RFC (ATAD5-RFC in humans) excludes DNA from its chamber and functions as a PCNA unloader. Together, these mechanisms illustrate how clamp loaders are diversified to accommodate DNA structure and replisome context, ensuring coordinated control of genome replication and maintenance.

9-1-1 clamp↗

PubMed2005

Bacillus subtilis bacteriophage SPP1-encoded gene 34.1 product is a recombination-dependent DNA replication protein.

SPP1-encoded replication and recombination proteins, involved in the early steps of the initiation of concatemeric DNA synthesis, have been analyzed. Dimeric G34.1P exonuclease degrades, with a 5' to 3' polarity and in a Mg2+-dependent reaction, preferentially linear double-stranded (ds) DNA rather than single-stranded (ss) DNA. Binding of the replisome organizer, G38P, to its cognate sites (oriDNA) halts the 5' to 3' exonucleolytic activity of G34.1P on dsDNA. The G35P recombinase increases the affinity of G34.1P for dsDNA, and stimulates G34.1P activity on dsDNA, but not on ssDNA. Then, filamented G35P promotes limited strand exchange with a homologous sequence. The ssDNA binding protein, G36P, protects ssDNA from the G34.1P exonuclease activity and stimulates G35P-catalyzed strand exchange. The data presented suggest a model for the role of G34.1P during initiation of sigma replication: G38P bound to oriDNA might halt replication fork progression, and G35P, G34.1P and G36P in concert might lead to the re-establishment of a unidirectional recombination-dependent replication that accounts for the direction of DNA packaging.

Adenosine Triphosphate↗

PubMed2006

Chromosome organization and segregation in bacteria.

In the recent years, considerable advances have been made towards understanding the structure and function of the bacterial chromosome. A number of different factors appear to cooperate in condensing DNA into a highly dynamic assembly of supercoiled loops. Despite this variability in the lower levels of chromatin structure, the global arrangement of chromosomal DNA within the cell is surprisingly conserved, with loci being arrayed along the cellular long axis in line with their order on the genomic map. This conserved pattern is propagated during the course of DNA segregation. First, after entry into S-phase, the newly synthesized origin regions are segregated in an active and directed process, involving the bacterial actin homolog MreB. Subsequent DNA segments then follow by different mechanisms. They are separated immediately after release from the replisome and move rapidly to their conserved positions in the incipient daughter cell compartments. Partitioning of the bacterial chromosome thus takes place while DNA replication is in progress.

Bacteria↗

PubMed2005

Biochemical and genetic analysis of the distinct proliferating cell nuclear antigens of Toxoplasma gondii.

The apicomplexa parasite Toxoplasma gondii expresses two distinct proliferating cell nuclear antigens (PCNA) that exhibit distinct patterns of subcellular localization during tachyzoite growth. In all cell cycle phases, TgPCNA1 is concentrated in the nucleus, while TgPCNA2 is only concentrated in the nucleus during S-phase and uniformly distributed throughout the cell during mitosis and early G1-phase. TgPCNA1-GFP and native TgPCNA2 display a punctate staining pattern that is consistent with assembly into replication foci during S-phase; however, TgPCNA2 disassociates from replication foci before TgPCNA1-GFP. Consistent with the distinct pattern of TgPCNA2 cellular localization, homotypic TgPCNA2 interactions were primarily observed by yeast two-hybrid or co-immunoprecipitation analysis. Transgenic parasites in which the TgPCNA2 gene was disrupted displayed a slower growth rate in vitro; however, no difference in DNA polymerase activity, response to chemical mutagens, or recombinational frequency was observed in these mutant clones demonstrating that TgPCNA2 is non-essential in the tachyzoite developmental stage. Heterologous expression of TgPCNA1, but not TgPCNA2, was able to complement a POL30 cold-sensitive yeast strain suggesting that this isoform may serve as a major replisomal factor in T. gondii and is consistent with the failure to disrupt this gene in tachyzoites.

Animals↗

PubMed2005

Exo1 processes stalled replication forks and counteracts fork reversal in checkpoint-defective cells.

The replication checkpoint coordinates the cell cycle with DNA replication and recombination, preventing genome instability and cancer. The budding yeast Rad53 checkpoint kinase stabilizes stalled forks and replisome-fork complexes, thus preventing the accumulation of ss-DNA regions and reversed forks at collapsed forks. We searched for factors involved in the processing of stalled forks in HU-treated rad53 cells. Using the neutral-neutral two-dimensional electrophoresis technique (2D gel) and psoralen crosslinking combined with electron microscopy (EM), we found that the Exo1 exonuclease is recruited to stalled forks and, in rad53 mutants, counteracts reversed fork accumulation by generating ss-DNA intermediates. Hence, Exo1-mediated fork processing resembles the action of E. coli RecJ nuclease at damaged forks. Fork stability and replication restart are influenced by both DNA polymerase-fork association and Exo1-mediated processing. We suggest that Exo1 counteracts fork reversal by resecting newly synthesized chains and resolving the sister chromatid junctions that cause regression of collapsed forks.

Cell Cycle↗

PubMed2006

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.

Bacteriophage T4↗

PubMed2006

DNA repeat rearrangements mediated by DnaK-dependent replication fork repair.

We propose that rearrangements between short tandem repeated sequences occur by errors made during a replication fork repair pathway involving a replication template switch. We provide evidence here that the DnaK chaperone of E. coli controls this template switch repair process. Mutants in dnaK are sensitive to replication fork damage and exhibit high expression of the SOS response, indicative of repair deficiency. Deletion and expansion of tandem repeats that occur by replication misalignment ("slippage") are also DnaK dependent. Because mutations in dnaX encoding the gamma and tau subunits of DNA polymerase III mimic dnaK phenotypes and are genetically epistatic, we propose that the DnaKJ chaperone remodels the replisome to facilitate repair. The fork remains largely intact because PriA or PriC restart proteins are not required. We also suggest that the poorly defined RAD6-RAD18-RAD5 mechanism of postreplication repair in eukaryotes occurs by an analogous mechanism to the DnaK template-switch pathway in prokaryotes.

DNA Polymerase III↗

PubMed2006

Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

Amino Acid Motifs↗

PubMed2003

Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep).

Geminiviruses replicate their small, single-stranded DNA genomes in plant nuclei using host replication machinery. Similar to most dicotyledonous plant-infecting geminiviruses, Tomato yellow leaf curl Sardinia virus (TYLCSV) encodes a protein, REn, that enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that REn protein from another geminivirus, Tomato golden mosaic virus (TGMV), forms oligomers and interacts with Rep protein, the only viral protein essential for replication. It has been shown that both proteins from TGMV also interact with a plant homolog of the mammalian tumor suppressor retinoblastoma protein (RBR). By using yeast two-hybrid technology and the TYLCSV REn protein as bait, we have isolated three clones of the proliferating cell nuclear antigen (PCNA) of Arabidopsis thaliana, a ring-shaped protein that encircles DNA and plays an essential role in eukaryotic chromosomal DNA replication. We also demonstrate by the two-hybrid system and a pull-down assay that REn interacts with tomato PCNA (LePCNA). Analysis of truncated proteins has located the REn-binding domain of LePCNA between amino acids 132 and 187, whereas all REn deletions used abolished or decreased dramatically its ability to interact with PCNA. Tomato PCNA also interacts with TYLCSV Rep. We propose that the interaction between PCNA and REn/Rep takes place during virus infection, inducing the assembly of the plant replication complex (replisome) close to the virus origin of replication.

Amino Acid Sequence↗

PubMed1996

Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement.

The E. coli replication fork synthesizes DNA at the rate of nearly 1000 nt/s. We show here that an interaction between the tau subunit of the replicative polymerase (the DNA polymerase III holoenzyme) and the replication fork DNA helicase (DnaB) is required to mediate this high rate of replication fork movement. In the absence of this interaction, the polymerase follows behind the helicase at a rate equal to the slow (approximately 35 nt/s) unwinding rate of the helicase alone, whereas upon establishing a tau-DnaB contact, DnaB becomes a more effective helicase, increasing its translocation rate by more than 10-fold. This finding establishes the existence of both a physical and communications link between the two major replication machines in the replisome: the DNA polymerase and the primosome.

Bacterial Proteins↗

PubMed2000

Sliding clamps: a (tail)ored fit.

New structural information on the architecture of a DNA replisome provides insights into a number of DNA metabolic processes and their modulation by circular 'sliding damps', which form rings around DNA that play an Important role in processive processes such as replication.

Animals↗

PubMed2000

PriA-directed replication fork restart in Escherichia coli.

The encounter of a replication fork with either a damaged DNA template, a nick in the template strand or a 'frozen' protein-DNA complex can stall the replisome and cause it to fall apart. Such an event generates a requirement for replication fork restart if the cell is going to survive. Recent evidence shows that replication fork restart is effected by the action of the recombination proteins generating a substrate for PriA-directed replication fork assembly.

Chromosomes, Bacterial↗

PubMed2001

Intricacies in ATP-dependent clamp loading: variations across replication systems.

DNA replication requires the coordinated effort of many proteins to create a highly processive biomachine able to replicate entire genomes in a single process. The clamp proteins confer on replisomes this property of processivity but in turn require clamp loaders for their functional assembly onto DNA. A more detailed view of the mechanisms for holoenzyme assembly in replication systems has been obtained from the advent of novel solution experiments and the appearance of low- and high-resolution structures for the clamp loaders.

Adenosine Triphosphate↗

PubMed1998

Thionucleobases as intrinsic photoaffinity probes of nucleic acid structure and nucleic acid-protein interactions.

In the past few years thionucleobases have been extensively used as intrinsic photolabels to probe the structure in solution of folded RNA molecules and to identify contacts within nucleic acids and/or between nucleic acids and proteins, in complex nucleoprotein assemblies. These thio residues such as 4-thiouracil found in E. coli tRNA and its non-natural congeners 4-thiothymine, 6-thioguanine and 6-mercaptopurine absorb light at wavelengths longer than 320 nm and, thus, can be selectively photoactivated. Synthetic or enzymatic procedures have been established, allowing the random or site-specific incorporation of thionucleotide(s) within a RNA (DNA) chain which, in most cases, retains unaltered structural and biological properties. Owing to the high photoreactivity of their triplet state (intersystem yield close to unity), 4-thiouracil and 4-thiothymine derivatives exhibit a high photocrosslinking ability towards pyrimidines (particularly thymine) but also purines. From the nature of the photoproducts obtained in base or nucleotide mixtures and in dinucleotides, the main photochemical pathway was identified as a (2 + 2) photoaddition of the excited C-S bond onto the 5, 6 double bond of pyrimidines yielding thietane intermediates whose structure could be characterized. Depending on the mutual orientation of these bonds in the thietanes, their subsequent dark rearrangement yielded, respectively, either the 5-4 or 6-4 bipyrimidine photoadduct. A similar mechanism appears to be involved in the formation of the unique photoadduct formed between 4-thiothymidine and adenosine. The higher reactivity of thymine derived acceptors can be explained by an additional pathway which involves hydrogen abstraction from the thymine methyl group, followed by radical recombination, leading to methylene linked bipyrimidines. The high photocrosslinking potential of thionucleosides inserted in nucleic acid chains has been used to probe RNA-RNA contacts within the ribosome permitting, in particular, the elucidation of the path of mRNA throughout the small ribosomal subunit. Functional interactions between the mRNA spliced sites and U RNAs could be detected within the spliceosome. Analysis of the photocrosslinks obtained within small endonucleolytic ribozymes in solution led to a tertiary folded pseudo-knot structure for the HDV ribozyme and allowed the construction of a Y form of a hammerhead ribozyme, which revealed to be in close agreement with the structure observed in crystals. Thionucleosides incorporated in nucleic acids crosslink efficiently amino-acid residues of proteins in contact with them. Despite the fact that little is known about the nature of the photoadducts formed, this approach has been extensively used to identify protein components interacting at a defined nucleic acid site and applied to various systems (replisome, spliceosome, transcription complexes and ribosomes).

Animals↗

PubMed1998

Coordinated leading and lagging strand DNA synthesis on a minicircular template.

The coordinated synthesis of both leading and lagging DNA strands is thought to involve a dimeric DNA polymerase and a looping of the lagging strand so that both strands can be synthesized in the same direction. We have constructed a minicircle with a replication fork that permits an assessment of the stoichiometry of the proteins and a measurement of the synthesis of each strand. The replisome consisting of bacteriophage T7 DNA polymerase, helicase, primase, and single-stranded DNA-binding protein mediates coordinated replication. The criteria for coordination are fulfilled: (1) a replication loop is formed, (2) leading and lagging strand synthesis are coupled, (3) the lagging strand polymerase recycles from one Okazaki fragment to another, and (4) the length of Okazaki fragments is regulated. T7 single-stranded DNA-binding protein is essential for coordination.

Bacteriophage T7↗