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Solution structure of the bacteriophage T4 DNA polymerase holoenzyme. Protein-protein interactions characterized on this work as well as previous investigations (7, 13–17, 32–33, 42) were used to build this model.
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Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as
intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to
the ground-state structures of the individual proteins. By using a variety of solution-phase technique...
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The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in whic...
Citations
... Whenever tandem MS spectra are obtained, their interpretation is far from trivial due to the population of these spectra with fragments from two different peptides. A number of strategies, such as isotope labeling methods either on the crosslinker [4,15,16] or at the Ctermini of peptides [17,18], crosslinking reagents with an affinity functionality [12,[19][20][21][22][23], and protein interaction reporter (PIR) technology in which upon tandem MS/MS fragmentation chemical crosslinking reagents can produce characteristic reporter ions to differentiate covalently linked peptides from other species [24][25][26], were developed to facilitate the identification of covalently linked peptides. Despite impressive progress in this direction, a move to more complex protein samples has been challenging as increases in protein size and number are paralleled by similar increases in the complexity of mass spectra. ...
The low resolution three-dimensional structure of a protein can be inferred from existing disulfide bridges or experimentally introduced chemical crosslinks. The general procedure involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently-linked peptides, native disulfide-linked peptides and chemically cross-linked peptides. To facilitate unambiguous identification of these peptides, an isocratic purification method was developed for selective enrichment of covalently-linked cyanogen bromide (CNBr) fragments. This method capitalizes on the ability of homoserine lactone moieties at the C-termini of CNBr cleavage products for selective conjugation of primary-amine containing affinity tag. The availability of two C-termini within covalently-linked peptides allows for the conjugation of two affinity tags, whereas the other peptides have only one affinity tag at the C-terminus, which enables selective enrichment of covalently-linked peptides by utilization of affinity tag with moderate dissociation constant. Here we demonstrate successful implementation of this method with tetrahistidine as the affinity tag for enrichment of covalently-linked CNBr fragments of test peptides and proteins.
... Single cysteine polymerases allow the attachment of fluorophores (and other probes) at defined positions, which is useful in numerous kinetic and single molecule experiments and proteinprotein interaction studies in the replisome. [17] Polymerases containing a sole cysteine could be useful for applications that require protein immobilisation, such as single molecule real time DNA sequencing. [18] Recently, the polymerase from the archaeon Thermococcus literalis was rendered uracil insensitive by chemical modification of an engineered cysteine with a bulky group. ...
The family-B DNA polymerases obtained from the order Thermococcales, for example, Pyrococcus furiosus (Pfu-Pol) are commonly used in the polymerase chain reaction (PCR) because of their high thermostability and low error rates. Most of these polymerases contain four cysteines, arranged as two disulfide bridges. With Pfu-Pol C429-C443 forms one of the disulfides (DB1) and C507-C510 (DB2) the other. Although the disulfides are well conserved in the enzymes from the hyperthermophilic Thermococcales, they are less prevalent in euryarchaeal polymerases from other orders, and tend to be only found in other hyperthermophiles. Here, we report on the effects of deleting the disulfide bridges by mutating the relevant cysteines to serines. A variety of techniques, including differential scanning calorimetry and differential scanning fluorimetry, have shown that both disulfides make a contribution to thermostability, with DB1 being more important than DB2. However, even when both disulfides are removed, sufficient thermostability remains for normal (identical to the wild type) performance in PCR and quantitative (real-time) PCR. Therefore, polymerases totally lacking cysteine are fully compatible with most PCR-based applications. This observation opens the way to further engineering of polymerases by introduction of a single cysteine followed by appropriate chemical modification.
... For this purpose, crosslinkers can be equipped with a functional group such as a biotin moiety which facilitate enrichment following their conjugation to a peptide. 24,[38][39][40][41][42][43] However, the application of more complex crosslinkers may be counterintuitive if the long-term objective is the study of protein complex topologies following in vivo crosslinking of cells and intact tissues. Furthermore, the use of affinity-tagged crosslinking reagents will lead to the concomitant purification of noninformative peptides which are merely derivatized or contain internal covalent linkages. ...
The low resolution structure of a protein can sometimes be inferred from information about existing disulfide bridges or experimentally introduced chemical crosslinks. Frequently, this task involves enzymatic digestion of a protein followed by mass spectrometry-based identification of covalently linked peptides. To facilitate this task, we developed a method for the enrichment of covalently linked peptides following the chemical cleavage of a protein. The method capitalizes on the availability of homoserine lactone moieties at the C-termini of cyanogen bromide cleavage products which support selective conjugation of affinity tags. The availability of two C-termini within covalently linked peptides allows for the conjugation of two distinct affinity tags and thereby enables subsequent removal of unmodified peptides by tandem affinity chromatography. Here, we demonstrate the stepwise implementation of this method using a polyhistidine tag and a biotin tag for the selective two-step purification of covalently linked cyanogen bromide fragments from increasingly complex protein samples. The method is independent of the nature of the covalent bond, is adaptable to fully denaturing conditions, and requires only low picomole quantities of starting material.
... Other possible pathways of assembling gp43 and gp45 on DNA have been proposed (72). T4 DNA polymerase uses its C-terminal region to interact with the trimeric clamp gp45 at the subunit interface (73,74). In contrast to E. coli, the dissociated clamp loader does not remain bound to the polymerase-clamp and primer-template complexes (71, 75) (compare Figures 3 and 4). ...
Replisomes are the protein assemblies that replicate DNA. They function as molecular motors to catalyze template-mediated polymerization of nucleotides, unwinding of DNA, the synthesis of RNA primers, and the assembly of proteins on DNA. The replisome of bacteriophage T7 contains a minimum of proteins, thus facilitating its study. This review describes the molecular motors and coordination of their activities, with emphasis on the T7 replisome. Nucleotide selection, movement of the polymerase, binding of the processivity factor, unwinding of DNA, and RNA primer synthesis all require conformational changes and protein contacts. Lagging-strand synthesis is mediated via a replication loop whose formation and resolution is dictated by switches to yield Okazaki fragments of discrete size. Both strands are synthesized at identical rates, controlled by a molecular brake that halts leading-strand synthesis during primer synthesis. The helicase serves as a reservoir for polymerases that can initiate DNA synthesis at the replication fork. We comment on the differences in other systems where applicable.
... Protein concentration of whole cell lysates was determined using a micro BCA assay kit (Pierce, Rockford, IL). Protein samples (40 μg total protein/sample) were subjected to SDS-PAGE separation and Western blot analysis as described (44,45). A rabbit polyclonal anti-TTP antibody (CARM3) was used at a 1:5000 dilution (starting concentration of 0.9 mg/mL) to detect TTP, while expression of the glucocorticoid receptor (GR) was tested using rabbit polyclonal anti-GR antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:500 dilution. ...
Glucocorticoids (GCs) are the mainstay of anti-inflammatory therapy. Modulation of posttranscriptional regulation (PTR) of gene expression by GCs is a relevant yet poorly characterized mechanism of their action. The RNA-binding protein tristetraprolin (TTP) plays a central role in PTR by binding to AU-rich elements in the 3'-untranslated region of proinflammatory transcripts and accelerating their decay. We found that GCs induce TTP expression in primary and immortalized human bronchial epithelial cells. To investigate the importance of PTR and the role of TTP in GC function, we compared the effect of GC treatment on genome-wide gene expression using mouse embryonic fibroblasts (MEFs) obtained from wild-type and TTP(-/-) mice. We confirmed that GCs induce TTP in MEFs and observed in TTP(-/-) MEFs a striking loss of up to 85% of GC-mediated gene expression. Gene regulation by TNF-alpha was similarly affected, as was the antagonistic effect of GC on TNF-alpha-induced response. Inflammatory genes, including cytokines and chemokines, were among the genes whose sensitivity to GCs was affected by lack of TTP. Silencing of TTP in WT MEFs by small interfering RNA confirmed loss of GC response in selected targets. Immunoprecipitation of ribonucleoprotein complexes revealed binding of TTP to several validated transcripts. Changes in the rate of transcript degradation studied by actinomycin D were documented for only a subset of transcripts bound to TTP. These results reveal a strong and previously unrecognized contribution of PTR to the anti-inflammatory action of GCs and point at TTP as a key factor mediating this process through a complex mechanism of action.
... 6 and 7). T4 DNA poly-merase, which catalyzes DNA synthesis on both leading and lagging strands, is attached to a sliding clamp protein (gene 45), loaded by the complex of the gene 44 and 62 proteins ( Fig. 1) (8,9). Gene 41 helicase moves 5Ј to 3Ј on the lagging strand template (10), opening the duplex ahead of the leading strand polymerase and interacting with the primase to allow it to make the RNA primers that initiate lagging strand synthesis (11)(12)(13)(14). ...
Our previous electron microscopy of DNA replicated by the bacteriophage T4 proteins showed a single complex at the fork, thought to contain the leading and lagging strand proteins, as well as the protein-covered single-stranded DNA on the lagging strand folded into a compact structure. "Trombone" loops formed from nascent lagging strand fragments were present on a majority of the replicating molecules (Chastain, P., Makhov, A. M., Nossal, N. G., and Griffith, J. D. (2003) J. Biol. Chem. 278, 21276-21285). Here we probe the composition of this replication complex using nanoscale DNA biopointers to show the location of biotin-tagged replication proteins. We find that a large fraction of the molecules with a trombone loop had two pointers to polymerase, providing strong evidence that the leading and lagging strand polymerases are together in the replication complex. 6% of the molecules had two loops, and 31% of these had three pointers to biotin-tagged polymerase, suggesting that the two loops result from two fragments that are being extended simultaneously. Under fixation conditions that extend the lagging strand, occasional molecules show two nascent lagging strand fragments, each being elongated by a biotin-tagged polymerase. T4 41 helicase is present in the complex on a large fraction of actively replicating molecules but on a smaller fraction of molecules with a stalled polymerase. Unexpectedly, we found that 59 helicase-loading protein remains on the fork after loading the helicase and is present on molecules with extensive replication.
... Solution-based studies have shown that one of the subunit interfaces in gp45 is in an open conformation (Alley et al., 1999b;Millar et al., 2004). In the presence of the bacteriophage T4 DNA polymerase, the gp45 clamp remains stably bound to DNA, because the C-terminal tail of the polymerase binds in the open interface to effectively close the ring (Alley et al., 1999a;Alley et al., 2001;Capson et al., 1991). ...
... FRET measurements have proved to be an extremely valuable tool for measuring dynamic and transient interactions that occur during the clamp loading reaction cycle (Alley et al., 2000;Alley et al., 1999b;Millar et al., 2004;Soumillion et al., 1998;Trakselis et al., 2001a;Trakselis et al., 2003) and between different components in the bacteriophage T4 replisome (Alley et al., 2001;Xi et al., 2005a;Xi et al., 2005b;Zhang et al., 2005). In the bacteriophage T4 clamp-loading reaction, FRET has been particularly useful in defining the stages at which the gp45 clamp opens and closes. ...
... Following DNA binding, hydrolysis of two more molecules of ATP is required to close the clamp, but the clamp is not closed completely (Alley et al., 2000;Sexton et al., 1998;Trakselis et al., 2001a;Trakselis et al., 2003). The bacteriophage T4 DNA polymerase ultimately binds the clamp in a manner in which the C-terminus is bound in the small opening left at the interface between clamp subunits to effectively close the ring (Alley et al., 1999a;Alley et al., 2001;Trakselis et al., 2001a). ...
Sliding clamps and clamp loaders are processivity factors required for efficient DNA replication. Sliding clamps are ring-shaped complexes that tether DNA polymerases to DNA to increase the processivity of synthesis. Clamp loaders assemble these ring-shaped clamps onto DNA in an ATP-dependent reaction. The overall process of clamp loading is dynamic in that protein-protein and protein-DNA interactions must actively change in a coordinated fashion to complete the mechanical clamp-loading reaction cycle. The clamp loader must initially have a high affinity for both the clamp and DNA to bring these macromolecules together, but then must release the clamp on DNA for synthesis to begin. Evidence is presented for a mechanism in which the clamp-loading reaction comprises a series of binding reactions to ATP, the clamp, DNA, and ADP, each of which promotes some change in the conformation of the clamp loader that alters interactions with the next component of the pathway. These changes in interactions must be rapid enough to allow the clamp loader to keep pace with replication fork movement. This review focuses on the measurement of dynamic and transient interactions required to assemble the Escherichia coli sliding clamp on DNA.
... The polymerase physically interacts with gp32, which has important consequences for the properties of gp43, such as higher affinity for the primer-template junction and increased processivity during DNA synthesis (21). The polymerase is in turn attached to the clamp protein through a well described interaction between the C terminus of the polymerase and the subunit interfaces of the trimeric clamp protein (5,6,22). Based on functional evidence, an additional interaction between gp41 helicase and the polymerase has been proposed (23,24). ...
The T4 helicase loading protein (gp59) interacts with a multitude of DNA replication proteins. In an effort to determine the functional consequences of these protein-protein interactions, point mutations were introduced into the gp59 protein. Mutations were chosen based on the available crystal structure and focused on hydrophobic residues with a high degree of solvent accessibility. Characterization of the mutant proteins revealed a single mutation, Y122A, which is defective in polymerase binding and has weakened affinity for the helicase. The interaction between single-stranded DNA-binding protein and Y122A is unaffected, as is the affinity of Y122A for DNA substrates. When standard concentrations of helicase are employed, Y122A is unable to productively load the helicase onto forked DNA substrates. As a result of the loss of polymerase binding, Y122A cannot inhibit the polymerase during nucleotide idling or prevent it from removing the primer strand of a D-loop. However, Y122A is capable of inhibiting strand displacement synthesis by polymerase. The retention of strand displacement inhibition by Y122A, even in the absence of a gp59-polymerase interaction, indicates that there are two modes of polymerase inhibition by gp59. Inhibition of the polymerase activity only requires gp59 to bind to the replication fork, whereas inhibition of the exonuclease activity requires an interaction between the polymerase and gp59. The inability of Y122A to interact with both the polymerase and the helicase suggests a mechanism for polymerase unlocking by the helicase based on a direct competition between the helicase and polymerase for an overlapping binding site on gp59.
... In all of these structures, including that of the phage RB69 gene 45 clamp with the peptide from RB69 DNA polymerase (38), the peptide is found in the same location in a hydrophobic pocket on the interdomain loop. However, there is evidence that the clamp-interacting peptide from T4 polymerase can also bind at the subunit interface of the 45 clamp in solution (49). When the structure of T4 RNase H was docked on the phage RB69 clamp by putting its N-terminal clamp interacting residues in the position of the RB69 DNA polymerase peptide, it was clear that the nuclease could not bind the clamp, if 32 protein was bound near the C terminus (not shown). ...
In the bacteriophage T4 DNA replication system, T4 RNase H removes the RNA primers and some adjacent DNA before the lagging strand fragments are ligated. This 5'-nuclease has strong structural and functional similarity to the FEN1 nuclease family. We have shown previously that T4 32 protein binds DNA behind the nuclease and increases its processivity. Here we show that T4 RNase H with a C-terminal deletion (residues 278-305) retains its exonuclease activity but is no longer affected by 32 protein. T4 gene 45 replication clamp stimulates T4 RNase H on nicked or gapped substrates, where it can be loaded behind the nuclease, but does not increase its processivity. An N-terminal deletion (residues 2-10) of a conserved clamp interaction motif eliminates stimulation by the clamp. In the crystal structure of T4 RNase H, the binding sites for the clamp at the N terminus and for 32 protein at the C terminus are located close together, away from the catalytic site of the enzyme. By using mutant T4 RNase H with deletions in the binding site for either the clamp or 32 protein, we show that it is the interaction of T4 RNase H with 32 protein, rather than the clamp, that most affects the maturation of lagging strand fragments in the T4 replication system in vitro and T4 phage production in vivo.
... The polymerase C-terminal region was shown to interact with the interdomain loop of one of the clamp subunits. However, solution studies revealed a clamp structure with one open and two closed interfaces (20) and strongly argued for an interaction between the polymerase C terminus and the open clamp subunit interface (21,22). The x-ray data and its associated holoenzyme model which used the weaker interdomain binding site of gp45 was predicted, however, to play some role in holoenzyme assembly, DNA replication, translesion bypass, or other replication associated processes (19,21). ...
... However, solution studies revealed a clamp structure with one open and two closed interfaces (20) and strongly argued for an interaction between the polymerase C terminus and the open clamp subunit interface (21,22). The x-ray data and its associated holoenzyme model which used the weaker interdomain binding site of gp45 was predicted, however, to play some role in holoenzyme assembly, DNA replication, translesion bypass, or other replication associated processes (19,21). ...
The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of ≈0.0013 sec⁻¹ was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution/replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.
• DNA replication
• polymerase processivity
• polymerase exchange
