Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA.

Tinker, Rachel L.; Kassavetis, George A.; Geiduschek, E. Peter

doi:10.1002/j.1460-2075.1994.tb06867.x

Cited by 64 publications

(66 citation statements)

References 50 publications

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“…The result shows that ϳ50% more ␤ assembled onto the DNA in the absence of EBNA1 (1632 fmol of ␤ loaded per 833 fmol of 5Ј-unblocked template). A plausible explanation for this increased level of ␤ is the extra 18 bp available in the absence of EBNA1, which may accommodate another ␤ clamp, consistent with previous studies showing that multiple ␤ clamps accumulate on duplex DNA molecules (4,35). In summary, the result suggests that the ␥ complex does not require interaction with the ssDNA/dsDNA junction at the 5Ј-end of the primer to assemble ␤ onto DNA.…”

Section: Figsupporting

confidence: 86%

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

Yao¹,

Leu²,

Anjelkovic³

et al. 2000

Journal of Biological Chemistry

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The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ringshaped protein, the ␤ clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The ␤ clamp is assembled onto DNA by the multiprotein ␥ complex clamp loader that opens and closes the ␤ ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the ␥ complex assembles ␤ onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the ␥ complex does not assemble ␤ onto relaxed closed circular DNA even at low ionic strength. Hence, a 3-end is not required for clamp loading, but a singlestranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the ␥ complex exhibits polarity; it acts specifically at the 3-ssDNA/dsDNA junction to assemble ␤ onto the DNA. The ␥ complex can assemble ␤ onto a primed site as short as 10 nucleotides, corresponding to the width of the ␤ ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3 terminus prevents the clamp loading reaction, indicating that the ␥ complex and its associated ␤ clamp interact with ϳ14 -16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20 -22 bp from the 3 terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the ␥ complex on the duplex portion of the primed site for function with ␤. Interestingly, DNA secondary structure elements placed near the 3 terminus impose similar steric limits on the ␥ complex and polymerase action with ␤. The possible biological significance of these structural constraints is discussed.Escherichia coli DNA polymerase III holoenzyme (pol III 1 holoenzyme) is a highly processive multisubunit replicase (1). Processivity is conferred to the polymerase by the ␤ subunit (2, 3). The ␤ subunit is a ring-shaped protein that completely encircles DNA and slides along the duplex (4, 5). The ␤ ring endows the polymerase with high processivity by binding directly to it, continuously tethering it to the template during synthesis (4). The ␤ ring does not assemble onto DNA by itself; for this, it requires the clamp loading action of the ␥ complex. The ␥ complex is composed of five different proteins (␥, ␦, ␦Ј, , and ) (1). Upon binding ATP, the ␥ complex opens the ␤ ring and positions it around the primed template (7,8). Hydrolysis of two molecules of ATP results in closing the ring around DNA and dissociation of the ␥ complex from the clamp (7, 49).Following the release of the clamp loader from the clamp, the catalytic core polymerase subassembly (pol ...

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Section: Figsupporting

confidence: 86%

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

Yao¹,

Leu²,

Anjelkovic³

et al. 2000

Journal of Biological Chemistry

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“…Importantly, PCNA is a homotrimer and forms a toroidal ring structure in solution (38,73) which is able to slide along the DNA helix (65,66,84). These properties might account for the bidirectional propagation of nucleosomal arrays over relatively large distances away from various repair sites including lesions subject to NER (17) and single-strand breaks (Fig.…”

Section: Discussionmentioning

confidence: 99%

A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

Moggs

Grandi

Quivy

et al. 2000

Molecular and Cellular Biology

305

259

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Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA-CAF-1 interaction in the context of DNA damage processing and checkpoint control.Sensing and signaling the presence of DNA damage to the cell cycle checkpoint machinery is crucial for the maintenance of genomic integrity and the regulation of cell cycle progression (12,25,61,97). Checkpoints respond to DNA damage by halting cell cycle progression, providing time for DNA repair. This strategy avoids the replication and segregation of damaged chromosomes which could otherwise lead to genomic instability. DNA damage is caused by physical and chemical agents as well as normal cellular processes including DNA replication and oxidative stress. A variety of distinct DNA repair mechanisms involving lesion-specific DNA damage recognition proteins have been characterized in eukaryotic cells (reviewed in reference 15). The DNA damage checkpoint machinery may recognize structural perturbations in DNA and/or components of the DNA damage processing machinery during specific phases of the cell cycle. Yeast model systems have proven powerful in identifying components of mitotic DNA damage checkpoint pathways (5,37,43,71,97) which, by analogy with signal transduction pathways, consist of sensor, transducer, and effector molecules. Several checkpoint proteins have been proposed to be directly involved in DNA damage recognition based on their similarity to proteins involved in DNA metabolism, including a structural relative of a 3Ј-5Ј exonuclease (Saccharomyces cerevisiae Rad17 [Rad17 sc ]) and a replication factor C (RF-C)-like protein (Rad24 sc ). Protein kinases such as Mec1 sc and Rad53 sc appear to transduce signals from DNA damage sensors to the cell cycle machinery. Significant progress has been made in delineating the proteinprotein interactions and phosphorylation events occurring among some of these factors and their potential interfaces with DNA repair (96). However, the mol...

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“…The internal surface of the trimer contains six α helices, and each helix is present in a structural repeat in each PCNA monomer. Since RFC loads the PCNA trimer onto DNA, it topologically links the PCNA trimer to the DNA, allowing PCNA to track along the DNA (136). Most probably, RFC also functions to unload the PCNA when DNA synthesis is complete, much like the bacterial and phage T4 counterparts (137)(138)(139).…”

Section: Proliferating Cell Nuclear Antigen (Pcna)mentioning

confidence: 99%

The Dna Replication Fork in Eukaryotic Cells

Waga

Stillman

1998

Annu. Rev. Biochem.

738

653

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Replication of the two template strands at eukaryotic cell DNA replication forks is a highly coordinated process that ensures accurate and efficient genome duplication. Biochemical studies, principally of plasmid DNAs containing the Simian Virus 40 origin of DNA replication, and yeast genetic studies have uncovered the fundamental mechanisms of replication fork progression. At least two different DNA polymerases, a single-stranded DNA-binding protein, a clamp-loading complex, and a polymerase clamp combine to replicate DNA. Okazaki fragment synthesis involves a DNA polymerase-switching mechanism, and maturation occurs by the recruitment of specific nucleases, a helicase, and a ligase. The process of DNA replication is also coupled to cell-cycle progression and to DNA repair to maintain genome integrity. CONTENTS

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Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA.

Cited by 64 publications

References 50 publications

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

DNA Structure Requirements for the Escherichia coliγ Complex Clamp Loader and DNA Polymerase III Holoenzyme

A CAF-1–PCNA-Mediated Chromatin Assembly Pathway Triggered by Sensing DNA Damage

The Dna Replication Fork in Eukaryotic Cells

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