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Michael A Trakselis

Publications and source records attributed to Michael A Trakselis.

12 recordsLinked to original sources
PubMed2005

Organization of the archaeal MCM complex on DNA and implications for the helicase mechanism.

The homomultimeric archaeal mini-chromosome maintenance (MCM) complex serves as a simple model for the analogous heterohexameric eukaryotic complex. Here we investigate the organization and orientation of the MCM complex of the hyperthermophilic archaeon Sulfolobus solfataricus (Sso) on model DNA substrates. Sso MCM binds as a hexamer and slides on the end of a 3'-extended single-stranded DNA tail of a Y-shaped substrate; binding is oriented so that the motor domain of the protein faces duplex DNA. Two candidate beta-hairpin motifs within the MCM monomer have partially redundant roles in DNA binding. Notably, however, conserved basic residues within these motifs have nonequivalent roles in the helicase activity of MCM. On the basis of these findings, we propose a model for the mechanism of the helicase activity of MCM and note parallels with SV40 T antigen.

Adenosine Triphosphatases↗
PubMed2005

Architecture of the bacteriophage T4 primosome: electron microscopy studies of helicase (gp41) and primase (gp61).

Replication of DNA requires helicase and primase activities as part of a primosome assembly. In bacteriophage T4, helicase and primase are separate polypeptides for which little structural information is available and whose mechanism of association within the primosome is not yet understood. Three-dimensional structural information is provided here by means of reconstructions from electron microscopic images. Structures have been calculated for complexes of each of these proteins with ssDNA in the presence of MgATPgammaS. Both the helicase (gp41) and primase (gp61) complexes are asymmetric hexagonal rings. The gp41 structure suggests two distinct forms that have been termed "open" and "closed." The gp61 structure is clearly a six-membered ring, which may be a trimer of dimers or a traditional hexamer of monomers. This structure provides conclusive evidence for an oligomeric primase-to-ssDNA stoichiometry of 6:1.

Bacteriophage T4↗
PubMed2005

Assembly of the bacteriophage T4 primosome: single-molecule and ensemble studies.

Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5'-O-(3-thiotriphosphate) (MgATPgammaS) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41.DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the primosome on the lagging strand of DNA at a replication fork.

Adenosine Triphosphate↗
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↗
PubMed2004

The dynamic processivity of the T4 DNA polymerase during replication.

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 approximately 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.

Bacteriophage T4↗
PubMed2003

Dissociative properties of the proteins within the bacteriophage T4 replisome.

DNA replication is a highly processive and efficient process that involves the coordination of at least eight proteins to form the replisome in bacteriophage T4. Replication of DNA occurs in the 5' to 3' direction resulting in continuous replication on the leading strand and discontinuous replication on the lagging strand. A key question is how a continuous and discontinuous replication process is coordinated. One solution is to avoid having the completion of one Okazaki fragment to signal the start of the next but instead to have a key step such as priming proceed in parallel to lagging strand replication. Such a mechanism requires protein elements of the replisome to readily dissociate during the replication process. Protein trapping experiments were performed to test for dissociation of the clamp loader and primase from an active replisome in vitro whose template was both a small synthetic DNA minicircle and a larger DNA substrate. The primase, clamp, and clamp loader are found to dissociate from the replisome and are continuously recruited from solution. The effect of varying protein concentrations (dilution) on the size of Okazaki fragments supported the protein trapping results. These findings are in accord with previous results for the accessory proteins but, importantly now, identify the primase as dissociating from an active replisome. The recruitment of the primase from solution during DNA synthesis has also been found for Escherichia coli but not bacteriophage T7. The implications of these results for RNA priming and extension during the repetitive synthesis of Okazaki fragments are discussed.

Adenosine Triphosphatases↗
PubMed2003

The application of a minicircle substrate in the study of the coordinated T4 DNA replication.

A reconstituted in vitro bacteriophage T4 DNA replication system was studied on a synthetic 70-mer minicircle substrate. This substrate was designed so that dGMP and dCMP were exclusively incorporated into the leading and the lagging strand, respectively. This design allows the simultaneous and independent measurement of the leading and lagging strand synthesis. In this paper, we report our results on the characterization of the 70-mer minicircle substrate. We show here that the minicircle substrate supports coordinated leading and lagging strand synthesis under the experimental conditions employed. The rate of the leading strand fork movement was at an average of approximately 150 nucleotides/s. This rate decreased to less than 30 nucleotides/s when the helicase was omitted from the reaction. These results suggest that both the holoenzyme and the primosome can be simultaneously assembled onto the minicircle substrate. The lagging strand synthesized on this substrate is of an average of 1.5 kb, and the length of the Okazaki fragments increased with decreasing [rNTPs]. The proper response of the Okazaki fragment size toward the change of the priming signal further indicates a functional replisome assembled on the minicircle template. The effects of various protein components on the leading and lagging strand synthesis were also studied. The collective results indicate that coordinated strand synthesis only takes place within certain protein concentration ranges. The optimal protein levels of the proteins that constitute the T4 replisome generally bracket the concentrations of the same proteins in vivo. Omission of the primase has little effect on the rate of dNMP incorporation or the rate of the fork movement on the leading strand within the first 30 s of the reaction. This inhibition only becomes significant at later times of the reaction and may be associated with the accumulation of single-stranded DNA leading to the collapse of active replisomes.

Bacteriophage T4↗
PubMed2003

Examination of the role of the clamp-loader and ATP hydrolysis in the formation of the bacteriophage T4 polymerase holoenzyme.

Transient kinetic analyses further support the role of the clamp-loader in bacteriophage T4 as a catalyst which loads the clamp onto DNA through the sequential hydrolysis of two molecules of ATP before and after addition of DNA. Additional rapid-quench and pulse-chase experiments have documented this stoichiometry. The events of ATP hydrolysis have been related to the opening/closing of the clamp protein through fluorescence resonance energy transfer (FRET). In the absence of a hydrolysable form of ATP, the distance across the subunit interface of the clamp does not increase as measured by intramolecular FRET, suggesting gp45 cannot be loaded onto DNA. Therefore, ATP hydrolysis by the clamp-loader appears to open the clamp wide enough to encircle DNA easily. Two additional molecules of ATP then are hydrolyzed to close the clamp onto DNA. The presence of an intermolecular FRET signal indicated that the dissociation of the clamp-loader from this complex occurred after guiding the polymerase onto the correct face of the clamp bound to DNA. The final holoenzyme complex consists of the clamp, DNA, and the polymerase. Although this sequential assembly mechanism can be generally applied to most other replication systems studied to date, the specifics of ATP utilization seem to vary across replication systems.

Adenosine Triphosphate↗
PubMed2002

Protein-protein interactions in the bacteriophage T4 replisome. The leading strand holoenzyme is physically linked to the lagging strand holoenzyme and the primosome.

The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.

Bacteriophage T4↗
PubMedDate not supplied

Identification and mapping of protein-protein interactions by a combination of cross-linking, cleavage, and proteomics.

Protein-protein interactions are vital for almost all cellular functions, and many require the formation of multiprotein complexes. Identification of the macroscopic and microscopic protein interactions within these complexes is essential in understanding their mechanisms, both under physiologic as well as pathologic conditions. This review describes the current technology available to investigate interactions between proteins utilizing chemical cross-linking and site-directed cleavage reagents, outlining the necessary steps involved in identifying interacting proteins both in vitro and in vivo. Once interacting proteins are identified, more information about the architecture of the assemblies is necessary. Unique separation techniques coupled with cross-linking and mass spectrometry can now identify specific interaction sites and lead to the development of quaternary structural protein models. Furthermore, combination of these methods with proteomic approaches enables the identification and analysis of complex interactions in vivo. Finally, future directions in cross-linking methodologies are discussed.

Cross-Linking Reagents↗