Dynamics of DNA-tracking by two sliding-clamp proteins.

Fu, Tsu-Ju; Sanders, Glenn M.; O’Donnell, Michael; Geiduschek, E. Peter

doi:10.1002/j.1460-2075.1996.tb00814.x

Cited by 26 publications

(30 citation statements)

References 40 publications

(57 reference statements)

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“…However, this large footprint is likely explained by the ability of the ␥ complex to load multiple clamps onto DNA (4), thereby covering the duplex portion of the primed template with several ␤ rings. Studies such as this in the T4 system have demonstrated that a 660-bp stretch of duplex DNA can be protected from DNase I via packing the entire region with the T4 clamp, gp45 protein (50).…”

Section: Discussionmentioning

confidence: 98%

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: Discussionmentioning

confidence: 98%

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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“…All of these studies blocked the DNA substrate in an irreversible manner, by binding either avidin or an antibody to the DNA ends or by incorporating some type of DNA secondary structure at the DNA ends. The lac repressor-lac operator interaction has been extensively characterized and has been used previously to block various replication proteins from tracking along the DNA (49,52). We took advantage of this system as a means to reversibly block DNA ends during analysis of the interaction of the MSH2-MSH6 and MLH1-PMS1 complexes with DNA immobilized on an IAsys affinity biosensor.…”

Section: The Lac Repressor-lac Operator Interaction Provides a Reversmentioning

confidence: 99%

“…The lac repressor-lac operator system has been used previously in studies of replication proteins tracking along the DNA (49). Here this system was used in conjunction with an IAsys biosensor instrument from Thermo Affinity Sensors, which detects total internal reflectance and allows monitoring of binding and dissociation in real time, in order to develop a system for studying the ability of MMR proteins to move along the DNA.…”

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confidence: 99%

Analysis of the Interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 Complexes with DNA Using a Reversible DNA End-blocking System

Mendillo

Mazur²,

Kolodner

2005

Journal of Biological Chemistry

117

207

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The Lac repressor-operator interaction was used as a reversible DNA end-blocking system in conjunction with an IAsys biosensor instrument (Thermo Affinity Sensors), which detects total internal reflectance and allows monitoring of binding and dissociation in real time, in order to develop a system for studying the ability of mismatch repair proteins to move along the DNA. The MSH2-MSH6 complex bound to a mispaired base was found to be converted by ATP binding to a form that showed rapid sliding along the DNA and dissociation via the DNA ends and also showed slow, direct dissociation from the DNA. In contrast, the MSH2-MSH6 complex bound to a base pair containing DNA only showed direct dissociation from the DNA. The MLH1-PMS1 complex formed both mispair-dependent and mispair-independent ternary complexes with the MSH2-MSH6 complex on DNA. The mispair-independent ternary complexes were formed most efficiently on DNA molecules with free ends under conditions where ATP hydrolysis did not occur, and only exhibited direct dissociation from the DNA. The mispair-dependent ternary complexes were formed in the highest yield on DNA molecules with blocked ends, required ATP and magnesium for formation, and showed both dissociation via the DNA ends and direct dissociation from the DNA.Errors that occur during DNA replication result in base-base mismatches and small insertion/deletion mismatches that if left uncorrected are fixed in the DNA as mutations by subsequent rounds of DNA replication. The DNA mismatch repair (MMR) 1 system normally corrects such errors in the cell and is highly conserved from bacteria to humans (1-5). Defects in the system lead to increased rates of accumulation of mutations, and in humans, inherited and somatic defects in MMR result in increased development of cancer (6 -9). The mechanism of MMR is best understood in Escherichia coli (3)(4)(5). In E. coli the MutS protein, which appears to function as a homodimer, serves as the mispair recognition factor (10 -13). MutL, another homodimer, interacts with the MutS complex in the presence of a mispair and ATP and activates the endonucleolytic activity of MutH (11, 14 -20). MutH makes single strand breaks in the newly synthesized daughter strand at transiently unmethylated GATC sequences, allowing the unwinding of the DNA by UvrD coupled with the degradation of the error-containing strand by one of at least four redundant exonucleases (21, 22). Then DNA polymerase III holoenzyme can resynthesize the DNA strand leaving a nick that is subsequently sealed by DNA ligase (23). In eukaryotic cells, three different MutS homologs form two different heterodimeric complexes, called MSH2-MSH6 (MutS␣) and MSH2-MSH3 (MutS␤) that together recognize base-base mispairs and insertion/deletion loops (1, 2, 24 -35). Similarly, three different MutL homologs form two different heterodimeric complexes, called MLH1-PMS1 (MutL␣; PMS1 Saccharomyces cerevisiae is PMS2 in humans) and MLH1-MLH3, that function in MMR (36 -38). Like their bacterial homologs, the MSH and MLH complexe...

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“…Gp45, the processivity factor of the T4 DNA polymerase, is loaded onto DNA at primer-template junctions by its conjugate clamp loader, the T4 gp44/gp62 complex. Because the DNA-mounted state of gp45 is transient (Fu et al, 1996), a supply of loading sites must be maintained for continual reattachment, which in turn requires continued DNA synthesis. This is the basis of the dependence of active T4 late transcription on concurrent DNA replication in vivo.…”

Section: Introductionmentioning

confidence: 99%

Dissection of the Bacteriophage T4 Late Promoter Complex

Nechaev

Geiduschek

2008

Journal of Molecular Biology

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Activated transcription of the bacteriophage T4 late genes is generated by a mechanism that stands apart from the common modalities of transcriptional regulation: the activator gp45 is the viral replisome's sliding clamp; two sliding clamp-binding proteins, gp33 and gp55, replace the host RNA polymerase (RNAP) σ subunit. We have mutagenized, reconfigured and selectively disrupted individual interactions of the sliding clamp with gp33 and gp55 and have monitored effects on transcription. The C-terminal sliding clamp-binding epitopes of gp33 and gp55 are perfectly interchangeable, but the functions of these two RNAP-sliding clamp connections differ, with only the gp33-gp45 linkage essential for activation. Formation of transcription-ready promoter complexes by the sliding clamp-activated wild-type T4 RNAP resists competition by high concentrations of the polyanion heparin. This avid formation of promoter complexes requires both linkages of the T4 late RNAP to the sliding clamp. Preopening the promoter compensates for loss of the gp55-gp45 but not the gp33-gp45 linkage. We interpret these findings in relation to the common model of transcriptional initiation in bacteria and highlight the roles of individual interactions of the promoter complex.

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Dynamics of DNA-tracking by two sliding-clamp proteins.

Cited by 26 publications

References 40 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

Analysis of the Interaction between the Saccharomyces cerevisiae MSH2-MSH6 and MLH1-PMS1 Complexes with DNA Using a Reversible DNA End-blocking System

Dissection of the Bacteriophage T4 Late Promoter Complex

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