The central dogma of molecular biology explains the mechanism by which traits are transferred and preserved from generation to generation. Three important processes are involved in the central dogma, and these are composed of the following replication, transcription, and translation. Replication, the first step in the unidirectional process, requires the participation of primosomes in order to properly unwound, prime, and replicate the double-stranded DNA (deoxyribonucleic acid). This journal article investigates the molecular activities during DNA replication using bacteriophage T4 primosome and offers possible models of primosome behavior. The findings are also compared with the activity of bacteriophage T7 in order to assess the various helicase-primase properties of primosomes.
Background
A bacteriophage is a virus that infects bacterial cells. Replication, on the other hand, is the process wherein the double-stranded DNA is separated into single strands and each single strand is then replicated through the help of certain enzymes. In this study, bacteriophage T4 primosome was used to investigate the activity during DNA replication. The T4 primosome is composed of a hexameric helicase (gp41) and an oligomeric primase (gp 61) where the former is directly involved in the separation of the two strands in the leading strand of the DNA while the latter participates in the synthesis of primers in the lagging strand. The leading strand of the DNA has the 5 to 3 direction while the lagging strand proceeds in the 3 to 5 direction, which allows the production of Okazaki fragments (approximately 1 kilobase segments of the unwound DNA).
Usage of the T4 primosome results in the formulation of three possible models of DNA replication. Figure 1 reveals these models. The first models states that the helicase, the enzyme that catalyzes the separation of the double-stranded DNA, hinders the production of a new primer. The second model states that some primase, enzyme catalyzing the priming of the DNA, dissociate from the helicase in order to produce new primers. Lastly, the third model shows that the primosome remains attached to the DNA and the unwound segment forms a loop once the primer is transferred. Consequently, it can be deduced the the entire replication activity is stopped in the first model when the helicase pauses its activity while the second model allows a continuous replication events during the production of a new primer.
Fig. 1. The three models of T4 replisome activity during DNA replication
Key Experiments and Discussion
In order to investigate DNA replication, a DNA hairpin situated between a magnetic bead and a glass surface was used. Video microscopy was used to record the activity of the DNA hairpin which was tracked and manipulated using the magnetic force. Bar and line graphs were used to easily present the experimental findings. Fig. 2 reveals the set-up of this in vitro model.
From the aforementioned set-up, the following findings were discovered the synthesis of the primers are dependent on the concentration of the ribonucleoside triphosphates (rNTPs) stopping of the T4 primosome is not significantly related to primer production and must only be a function of DNA stability disassembling of the primosome can happen during the synthesis of the primer and loops can really be formed during priming. Formation of loops is found to be a new mechanism that enables the prevention of primase dissociation.Consequently, synthesis and utilization of primers is said to be a stochastic process that involves independent primer formation and helicase application. However, a different scenario was determined for T7 primosome because it allows the pausing of the entire replication process to allow the primer synthesis. Thus, results from this study shows that different helicase-primase activities co-exist in nature in order to favor maximum efficiency of the DNA replication process.
Fig. 2. Synthesis of primer and helicase activity as observed using experimental set-up. (a). The arrangement of the DNA hairpin and magnet, (b) graphical representation of the gp41 helicase activity (green line) and wild-type primosome (red line), (c)features of hairpin reannealing, (d) frequency of experimental replication processes (Manosas et al., 2009).
Background
A bacteriophage is a virus that infects bacterial cells. Replication, on the other hand, is the process wherein the double-stranded DNA is separated into single strands and each single strand is then replicated through the help of certain enzymes. In this study, bacteriophage T4 primosome was used to investigate the activity during DNA replication. The T4 primosome is composed of a hexameric helicase (gp41) and an oligomeric primase (gp 61) where the former is directly involved in the separation of the two strands in the leading strand of the DNA while the latter participates in the synthesis of primers in the lagging strand. The leading strand of the DNA has the 5 to 3 direction while the lagging strand proceeds in the 3 to 5 direction, which allows the production of Okazaki fragments (approximately 1 kilobase segments of the unwound DNA).
Usage of the T4 primosome results in the formulation of three possible models of DNA replication. Figure 1 reveals these models. The first models states that the helicase, the enzyme that catalyzes the separation of the double-stranded DNA, hinders the production of a new primer. The second model states that some primase, enzyme catalyzing the priming of the DNA, dissociate from the helicase in order to produce new primers. Lastly, the third model shows that the primosome remains attached to the DNA and the unwound segment forms a loop once the primer is transferred. Consequently, it can be deduced the the entire replication activity is stopped in the first model when the helicase pauses its activity while the second model allows a continuous replication events during the production of a new primer.
Fig. 1. The three models of T4 replisome activity during DNA replication
Key Experiments and Discussion
In order to investigate DNA replication, a DNA hairpin situated between a magnetic bead and a glass surface was used. Video microscopy was used to record the activity of the DNA hairpin which was tracked and manipulated using the magnetic force. Bar and line graphs were used to easily present the experimental findings. Fig. 2 reveals the set-up of this in vitro model.
From the aforementioned set-up, the following findings were discovered the synthesis of the primers are dependent on the concentration of the ribonucleoside triphosphates (rNTPs) stopping of the T4 primosome is not significantly related to primer production and must only be a function of DNA stability disassembling of the primosome can happen during the synthesis of the primer and loops can really be formed during priming. Formation of loops is found to be a new mechanism that enables the prevention of primase dissociation.Consequently, synthesis and utilization of primers is said to be a stochastic process that involves independent primer formation and helicase application. However, a different scenario was determined for T7 primosome because it allows the pausing of the entire replication process to allow the primer synthesis. Thus, results from this study shows that different helicase-primase activities co-exist in nature in order to favor maximum efficiency of the DNA replication process.
Fig. 2. Synthesis of primer and helicase activity as observed using experimental set-up. (a). The arrangement of the DNA hairpin and magnet, (b) graphical representation of the gp41 helicase activity (green line) and wild-type primosome (red line), (c)features of hairpin reannealing, (d) frequency of experimental replication processes (Manosas et al., 2009).
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