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PubMed2001

Visualization of mismatch repair in bacterial cells.

We determined the localizations of mismatch repair proteins in living Bacillus subtilis cells. MutS-GFP colocalized with the chromosome in all cells and formed foci in a subset of cells. MutL-GFP formed foci in a subset of cells, and its localization was MutS dependent. The introduction of mismatches by growth in 2-aminopurine caused a replication-dependent increase in the number of cells with MutS and MutL foci. Approximately half of the MutS foci colocalized with DNA polymerase foci. We conclude that MutS is associated with the entire chromosome, poised to detect mismatches. After detection, it appears that mismatch repair foci assemble at mismatches as they emerge from the DNA polymerase and are then carried away from the replisome by continuing replication.

2-Aminopurine↗
PubMed2000

Mechanisms of DNA replication.

Advances in recent years have led to exciting new ideas about the initiation, regulation and coordination of DNA replication. Structural studies have yielded fascinating glimpses of replisome action. In addition, the involvement of replication proteins in other cellular processes has blurred the lines between replication, repair and recombination.

DNA Replication↗
PubMed1982

Nuclear matrix-bound deoxyribonucleic acid synthesis: an in vitro system.

A nuclear matrix-bound in vitro replicational system that utilizes alpha-polymerase and matrix-attached DNA has been isolated from regenerating rat liver and characterized for in vitro requirements. beta-Polymerase is a minor component of the matrix in vitro system. Evidence is presented for the native association of alpha-polymerase with the nuclear matrix and for the parallel induction of matrix-bound alpha-polymerase and in vivo DNA synthesis during the proliferative response following partial hepatectomy. Our data suggest that the attachment of alpha-polymerase to the matrix may be an important step in the assembly of functional replisomes. We also demonstrate prereplicative and late-replicative rises in nuclear and matrix-bound beta-polymerase activity and discuss these results in terms of possible genome screening.

Animals↗
PubMed2004

Inhibition of protein interactions with the beta 2 sliding clamp of Escherichia coli DNA polymerase III by peptides from beta 2-binding proteins.

The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair.

Amino Acid Motifs↗
PubMed2004

On the solution structure of the T4 sliding clamp (gp45).

Examination by time-resolved fluorescence spectroscopy of the trimeric bacteriophage T4 clamp protein labeled across its three subunit interfaces with a fluorescence resonance energy transfer (FRET) pair indicates that the clamp exists in just one state in solution, with one open and two closed interfaces. This is in contrast to what is observed in the X-ray crystal structure. The population distribution of the trFRET distance is bimodal, giving 67% as 17 A and 33% as 42 A. This leads to the conclusion that gp45 exists in an asymmetric open state in solution. The further increase in the separation of the FRET pair in the presence of the clamp loader and ATP may be ascribed to either further opening of the open interface or the opening of a closed interface. The ramifications for replisome remodeling by this pathway are discussed.

Bacteriophage T4↗
PubMed2006

Single-molecule investigation of the T4 bacteriophage DNA polymerase holoenzyme: multiple pathways of holoenzyme formation.

In T4 bacteriophage, the DNA polymerase holoenzyme is responsible for accurate and processive DNA synthesis. The holoenzyme consists of DNA polymerase gp43 and clamp protein gp45. To form a productive holoenzyme complex, clamp loader protein gp44/62 is required for the loading of gp45, along with MgATP, and also for the subsequent binding of polymerase to the loaded clamp. Recently published evidence suggests that holoenzyme assembly in the T4 replisome may take place via more than one pathway [Zhuang, Z., Berdis, A. J., and Benkovic, S. J. (2006) Biochemistry 45, 7976-7989]. To demonstrate unequivocally whether there are multiple pathways leading to the formation of a productive holoenzyme, single-molecule fluorescence microscopy has been used to study the potential clamp loading and holoenzyme assembly pathways on a single-molecule DNA substrate. The results obtained reveal four pathways that foster the formation of a functional holoenzyme on DNA: (1) clamp loader-clamp complex binding to DNA followed by polymerase, (2) clamp loader binding to DNA followed by clamp and then polymerase, (3) clamp binding to DNA followed by clamp loader and then polymerase, and (4) polymerase binding to DNA followed by the clamp loader-clamp complex. In all cases, MgATP is required. The possible physiological significance of the various assembly pathways is discussed in the context of replication initiation and lagging strand synthesis during various stages of T4 phage replication.

Bacteriophage T4↗
PubMed2000

Metabolic functions of microbial nucleoside diphosphate kinases.

This article summarizes research from our laboratory on two aspects of the biochemistry of nucleoside diphosphate kinase from Escherichia coli--first, its interactions with several T4 bacteriophage-coded enzymes, as part of a multienzyme complex for deoxyribonucleoside triphosphate biosynthesis. We identify some of the specific interactions and discuss whether the complex is linked physically or functionally with the T4 DNA replication machinery, or replisome. Second, we discuss phenotypes of an E. coli mutant strain carrying a targeted deletion of ndk, the structural gene for nucleoside diphosphate kinase. How do bacteria lacking this essential housekeeping enzyme synthesize nucleoside triphosphates? In view of the specific interactions of nucleoside diphosphate kinase with T4 enzymes of DNA metabolism, how does T4 multiply after infection of this host? Finally, the ndk disruption strain has highly biased nucleoside triphosphate pools, including elevations of the CTP and dCTP pools of 7- and 23-fold, respectively. Accompanied by these biased nucleotide pools is a strong mutator phenotype. What is the biochemical basis for the pool abnormalities and what are the mutagenic mechanisms? We conclude with brief references to related work in other laboratories.

Bacteriophage T4↗
PubMed2003

A lack of telomeric non-reciprocal recombination (TENOR) may account for the premature proliferation blockade of Werner's syndrome fibroblasts.

Cell lineages survive as long as their descendants replicate. In human diploid fibroblasts (HDF) there is a telomere-driven clock that determines the limited replicative life span of their cell branches. Werner's syndrome fibroblasts (WF) prematurely limit their replicative life span compared to HDF on account of a defective RecQ helicase, WRNp. Here, we introduce the concept of telomeric non-reciprocal recombination (TENOR) and suggest that its dysregulation in WF is responsible for their premature senescence. We postulate that TENOR functions at the level of four-strand replisomes which are specific for and initiated at fused chromosome ends. Consequently, healthy replicatively senescent fibroblasts can undergo one further division mediated by a TENOR event. Thereby, one of the two daughter cells re-acquires proliferation capacity that initiates a new proliferating cell branch from the formerly replicatively senescent cell. We argue that the unique mechanisms of action of WRNp support this concept, and we apply it to WF. If it is stipulated that WF are defective for TENOR because of their defective WRNp, a simple model emerges as to how they become prematurely senescent.

Cell Division↗
PubMed2006

Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling.

In bacteria, the initiation of replication is controlled by DnaA, a member of the ATPases associated with various cellular activities (AAA+) protein superfamily. ATP binding allows DnaA to transition from a monomeric state into a large oligomeric complex that remodels replication origins, triggers duplex melting and facilitates replisome assembly. The crystal structure of AMP-PCP-bound DnaA reveals a right-handed superhelix defined by specific protein-ATP interactions. The observed quaternary structure of DnaA, along with topology footprint assays, indicates that a right-handed DNA wrap is formed around the initiation nucleoprotein complex. This model clarifies how DnaA engages and unwinds bacterial origins and suggests that additional, regulatory AAA+ proteins engage DnaA at filament ends. Eukaryotic and archaeal initiators also have the structural elements that promote open-helix formation, indicating that a spiral, open-ring AAA+ assembly forms the core element of initiators in all domains of life.

Adenosine Triphosphate↗
PubMed2004

Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance.

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.

Cell Cycle↗
PubMed2006

Distinct roles for Sld3 and GINS during establishment and progression of eukaryotic DNA replication forks.

The Cdc45 protein is crucial for the initiation of chromosome replication in eukaryotic cells, as it allows the activation of prereplication complexes (pre-RCs) that contain the MCM helicase. This causes the unwinding of origins and the establishment of DNA replication forks. The incorporation of Cdc45 at nascent forks is a highly regulated and poorly understood process that requires, in budding yeast, the Sld3 protein and the GINS complex. Previous studies suggested that Sld3 is also important for the progression of DNA replication forks after the initiation step, as are Cdc45 and GINS. In contrast, we show here that Sld3 does not move with DNA replication forks and only associates with MCM in an unstable manner before initiation. After the establishment of DNA replication forks from early origins, Sld3 is no longer essential for the completion of chromosome replication. Unlike Sld3, GINS is not required for the initial recruitment of Cdc45 to origins and instead is necessary for stable engagement of Cdc45 with the nascent replisome. Like Cdc45, GINS then associates stably with MCM during S-phase.

Cell Cycle Proteins↗
PubMed2006

Tracking of controlled Escherichia coli replication fork stalling and restart at repressor-bound DNA in vivo.

We report an efficient, controllable, site-specific replication roadblock that blocks cell proliferation, but which can be rapidly and efficiently reversed, leading to recovery of viability. Escherichia coli replication forks of both polarities stalled in vivo within the first 500 bp of a 10 kb repressor-bound array of operator DNA-binding sites. Controlled release of repressor binding led to rapid restart of the blocked replication fork without the participation of homologous recombination. Cytological tracking of fork stalling and restart showed that the replisome-associated SSB protein remains associated with the blocked fork for extended periods and that duplication of the fluorescent foci associated with the blocked operator array occurs immediately after restart, thereby demonstrating a lack of sister cohesion in the region of the array. Roadblocks positioned near oriC or the dif site did not prevent replication and segregation of the rest of the chromosome.

Cell Proliferation↗
PubMed2005

Slx4 becomes phosphorylated after DNA damage in a Mec1/Tel1-dependent manner and is required for repair of DNA alkylation damage.

Members of the RecQ family of DNA helicases, mutated in several syndromes associated with cancer predisposition, are key regulators of genome stability. The Saccharomyces cerevisiae SLX4 gene is required for cell viability in the absence of Sgs1, the only yeast RecQ helicase. SLX4 encodes one subunit of the heterodimeric Slx1-Slx4 endonuclease, although its cellular function is not clear. Slx1-Slx4 was reported to preferentially cleave replication fork-like structures in vitro, and cells lacking SLX4 are hypersensitive to DNA alkylation damage. Here we report that Slx4 becomes phosphorylated in cells exposed to a wide range of genotoxins. Even though it has been proposed that the role of Slx4 is restricted to S-phase, Slx4 phosphorylation is observed in cells arrested in G1 or G2 phases of the cell cycle, but not during an unperturbed cell cycle. Slx4 phosphorylation is completely abolished in cells lacking the Mec1 and Tel1 protein kinases, critical regulators of genome stability, but is barely affected in the absence of both Rad53 and Chk1 kinases. Finally we show that, whereas both Slx1 and Slx4 are dispensable for activation of cell-cycle checkpoints, Slx4, but not Slx1, is required for repair of DNA alkylation damage in both aynchronously growing cells and in G2-phase-arrested cells. These results reveal Slx4 as a new target of the Mec1/Tel1 kinases, with a crucial role in DNA repair that is not restricted to the processing of stalled replisomes.

Alkylation↗
PubMed2002

Fission yeast chk1 mutants show distinct responses to different types of DNA damaging treatments.

BACKGROUND: Chk1 kinase is activated by phosphorylation at serine-345 by Rad3 checkpoint kinase and is required for DNA damage checkpoint in late S and G2 phase of S. pombe cell cycle. We studied the ability of two chk1 mutants, chk1-1 and chk1-2, to undergo phosphorylation and to delay cell cycle progression in response to different types of DNA lesions. RESULTS: Both the Chk1-1 and Chk1-2 mutant proteins are phosphorylated to various extents when DNA is damaged in early G2 phase of cell cycle by either UV irradiation or gamma irradiation. However, chk1-2 mutant does not delay cell cycle progression in a dose dependent manner specifically upon gamma irradiations. This defect is not associated with an important loss of survival. Furthermore, both chk1 mutants survive to Camptothecin treatment despite undetectable Chk1-1 or Chk1-2 phosphorylated forms. We show that both mutant proteins are not phosphorylated in cds1 devoid cells treated with ribonucleotide reductase inhibitor hydroxyurea or when the replisome is affected by a thermosensitive mutation in DNA polymerase delta. This inability is associated with the loss of checkpoint function. We found that an increased level of Crb2/Rhp9 protein specifically complements the defect of the chk1-1 mutant allowing Chk1-1 phosphorylation upon treatment with hydroxyurea of dcds1 cells. CONCLUSIONS: Mutants chk1-1 and chk1-2 behave differently according to the type of lesion generated on DNA.

Alleles↗
PubMed2000

Subcellular localization of Dna-initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids.

We examined the intracellular distribution of Bacillus subtilis Dna-initiation proteins by immunofluorescence microscopy to visualize the initiation complex of replication in vivo. DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI were always detected as foci during the cell-division cycle. Interaction of DnaI with the DnaC helicase by the yeast two-hybrid assay suggests that DnaI acts as a helicase loader. The number of DnaB and DnaI foci within the cell exceeded that of oriC. Although the foci were not always co-localized with oriC, they seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication. When the replication cycle was synchronized in cells using a temperature-sensitive dnaA mutant, duplication of the oriC region was observed predominantly near an edge of the nucleoid. Before initiation occurred, each one of the DnaB and DnaI foci was frequently observed near there. Furthermore, DnaX-GFP (DnaX is a component of DNA polymerase III) foci were detected near either of the edges of the nucleoids at the onset of replication. These results suggest that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B. subtilis.

Animals↗
PubMed2000

Mid-cell Z ring assembly in the absence of entry into the elongation phase of the round of replication in bacteria: co-ordinating chromosome replication with cell division.

We have shown previously that, when spores of a thymine-requiring strain of Bacillus subtilis were grown out in the absence of thymine, mid-cell Z rings formed over the nucleoid and much earlier than might be expected with respect to progression into the round of replication. It is now shown that such conditions allow no replication of oriC. Rather than replication, partial degradation of the oriC region occurs, suggesting that the status of this region is connected with the 'premature' mid-cell Z ring assembly. A correlation was observed between entry into the replication elongation phase and a block to mid-cell Z rings. The conformation of the nucleoid under various conditions of DNA replication inhibition or limitation suggests that relief of nucleoid occlusion is not primarily responsible for mid-cell Z ring formation in the absence of thymine. We propose the existence of a specific structure at mid-cell that defines the Z ring nucleation site (NS). It is suggested that this NS is normally masked by the replisome upon initiation of replication or soon after entry into the elongation phase, and subsequently unmasked relatively late in the round. During spore outgrowth in the absence of thymine, this checkpoint control over mid-cell Z ring assembly breaks down prematurely.

Bacillus subtilis↗
PubMed2003

PriA supports two distinct pathways for replication restart in UV-irradiated Escherichia coli cells.

The Escherichia coli PriA protein loads the DnaB replicative helicase at branched DNA structures independently of the replication initiator protein, DnaA, and thereby facilitates assembly of functional replisomes at sites removed from oriC. It is therefore a critical factor in the rescue of replication forks stalled at DNA lesions. It is also a DNA helicase. We describe insertions near the 3' end of priA that interfere with PriA activity. These insertions and the previously described priA300 encoding helicase-defective PriA K230R are shown to be effective suppressors of the DNA repair defect in recG strains, but substantially reduce the ability of ruv mutants to survive DNA damage. The data presented suggest that PriA helicase in conjunction with RecG can promote direct rescue of stalled forks independently of the recombinational pathway promoted by the combined activities of the RuvABC, RecBCD and RecA proteins, which requires only the primosome assembly activity of PriA to load DnaB at D loops. In cells lacking the helicase activity of PriA, we propose that stalled forks can be redirected to the recombination pathway via a Holliday junction intermediate common to both pathways, thus explaining the resistance of these cells to DNA damage.

Adenosine Triphosphatases↗
PubMed2003

Replication restart in gyrB Escherichia coli mutants.

Gyrase is an essential topoisomerase in bacteria that introduces negative supercoils in DNA and relaxes the positive supercoils that form downstream of proteins tracking on DNA, such as DNA or RNA polymerases. Two gyrase mutants that suffer partial loss of function were used here to study the need for replication restart in conditions in which gyrase activity is affected. We show that the preprimosomal protein PriA is essential for the viability of these gyrB mutants. The helicase function of PriA is not essential. The lethality of the gyrB priA double mutants is suppressed by a dnaC809 mutation, indicating a requirement for primosome assembly in gyrB strains. The lethality of gyrB priA combination of mutations is independent of the level of DNA supercoiling, as gyrB and priA were also co-lethal in the presence of a DeltatopA mutation. Inactivation of homologous recombination did not affect the viability of gyrB mutants, indicating that replication restart does not require the formation of a recombination intermediate. We propose that the replisome is disassembled from replication forks when replication progression is blocked by the accumulation of positive supercoils in gyrase mutants, and that replication restarts via PriA-dependent primosome assembly, directly on the in-activated replication forks, without the formation of a recombination intermediate.

Adenosine Triphosphatases↗