Why is primase required for replication

These studies concluded that the Firmicutes are presumably among the most ancient bacteria and that the Aquificales have diverged much later in evolution [ 96 ]. Indeed, an analysis of the GC content of rRNA clusters suggests that hyperthermophilic species have evolved from mesophilic organisms via adaptation to high temperature [ 98 ] and that the Gram-negative double membrane may have been derived from sporulating Gram-positives [ 99 , ].

Thus, the study of C. All oligonucleotides and plasmids constructed for this study are listed in electronic supplementary material, tables S1 and S2. Construction of the plasmids for the bacterial two-hybrid system was performed with Gateway cloning technology Invitrogen.

Primers were generated with Primer X, a web-based tool for automatic design of mutagenic primers for site-directed mutagenesis. Overexpression of all proteins was carried out in E. Detailed description of the protein purifications is available as the electronic supplementary material. Below, we provide a short summary of the purification process. Protein concentrations mentioned in this manuscript refer to concentration of the monomer of the protein.

The elution profiles from each experiment were monitored at nm and plotted as a function of the elution volume. To assess interactions between CD and CD, purified proteins were mixed in a 1 : 1 stoichiometry based on monomer concentrations. Gel filtration standards Bio-Rad were run to calculate MW estimates.

Experiments were performed in triplicate. All reactions, containing 0. Proteins were added sequentially, with a 5 min preincubation after each addition. The gel was dried, scanned and analysed using a molecular imager and associated software Bio-Rad. Experiments were carried out in triplicate, and data analysis was performed using P rism 6 GraphPad Software. RNA priming assays and denaturing HPLC analyses were conducted as was previously described for other mesophilic bacterial primases [ 76 ].

For the denaturing HPLC analyses, a gradient 0—8. The moles of RNA primers synthesized were quantified as previously described [ 56 ]. Klose University of Texas at San Antonio. Jex and J. Gibson are acknowledged for exploratory helicase activity assays and analytical gel filtrations. We thank J. Tomkiewicz for help with preparing electronic supplementary material, figure S7. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

National Center for Biotechnology Information , U. Journal List Open Biol v. Open Biol. Published online Dec Friggen , 1 Marilynn A. Larson , 4, 5 Keith Spriggs , 3 Geoffrey S. Annemieke H.

Marilynn A. Geoffrey S. Author information Article notes Copyright and License information Disclaimer. Received Sep 28; Accepted Nov This article has been cited by other articles in PMC. Associated Data Supplementary Materials Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile - Smits et al.

Supporting information. Abstract DNA replication is an essential and conserved process in all domains of life and may serve as a target for the development of new antimicrobials. Keywords: DNA replication initiation, helicase loading and activation, primase trinucleotide specificity, ATPase, Clostridium difficile.

Background Extensive research, primarily on the model organisms Escherichia coli Gram- negative and Bacillus subtilis Gram-positive , has shown that many different proteins are involved in DNA replication. Results 2. In silico identification of putative replication initiation proteins In B. Open in a separate window.

Figure 1. Helicase can form hexamers at high concentration A distinguishing feature of the different modes of helicase loading ring-maker versus ring-breaker is the multimeric state of helicase at dilute concentrations of protein [ 6 ].

Figure 2. Mutation of the helicase Walker A motif abrogates protein—protein interactions To address the question of which of the proteins or whether both require ATP to promote the formation of a CDCD complex, mutants in the Walker A motif of both proteins were created.

Figure 3. Helicase loading of Clostridium difficile differs from Bacillus subtilis So far, our data show that the replicative helicase of C. Clostridium difficile helicase is activated by primase Primase has been shown to interact with helicase and stimulate its activity in a variety of organisms [ 16 , 57 , 61 ]. Figure 4. Clostridium difficile primase trinucleotide specificity is similar to Aquifex aeolicus primase Above, we have established a crucial role for the C.

Figure 5. A lysine residue contributes to trinucleotide specificity of primase Considering that the C. Figure 6. Discussion In silico analysis of the C. Material and methods 4. Plasmid construction and site-directed mutagenesis All oligonucleotides and plasmids constructed for this study are listed in electronic supplementary material, tables S1 and S2.

All constructs were verified by DNA sequencing. Purification of proteins Overexpression of all proteins was carried out in E. Supplementary Material Primase is required for helicase activity and helicase alters the specificity of primase in the enteropathogen Clostridium difficile - Smits et al.

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There are three possible mechanisms that can explain DNA's semiconservative replication. In the first mechanism one daughter strand is initiated at an origin on one parental strand and the second is initiated at another origin on the opposite parental strand. Thus only one strand grows from each origin. Some viruses use this type of mechanism.

The site at which the two strands are replicated is called the replication fork. Since the fork moves in one direction from the origin this type of replication is called unidirectional.

Some types of bacteria use this type of mechanism. This type of replication is called bi-directional. Most organisms, including mammals, use bi-directional replication. Requirements for DNA Synthesis. There are four basic components required to initiate and propagate DNA synthesis.

They are: substrates, template, primer and enzymes. Four deoxyribonucleotide triphosphates dNTP's are required for DNA synthesis note the only difference between deoxyribonucleotides and ribonucleotides is the absence of an OH group at position 2' on the ribose ring.

The high energy phosphate bond between the a and b phosphates is cleaved and the deoxynucleotide monophosphate is incorporated into the new DNA strand. The nucleotide that is to be incorporated into the growing DNA chain is selected by base pairing with the template strand of the DNA.

An enzyme, DNA polymerase, is required for the covalent joining of the incoming nucleotide to the primer. To actually initiate and sustain DNA replication requires many other proteins and enzymes which assemble into a large complex called a replisome.

It is thought that the DNA is spooled through the replisome and replicated as it passes through. The major catalytic step of DNA synthesis is shown below. Notice that DNA synthesis always occurs in a 5' to 3' direction and that the incoming nucleotide first base pairs with the template and is then linked to the nucleotide on the primer.