Linear Retroplasmid Replication Cycle

When the findings of the characterization studies of plasmid replication intermediates are combined with the properties of the pFOXC-RT revealed from in vitro reverse transcription assays, a model for linear clothespin retroplasmid replication can be developed (Fig. 4). Replication begins with transcription of the plasmid DNA, most likely by a host mitochondrial RNA polymerase. The length of the RNA transcripts suggests transcription likely begins on the top strand and proceeds around the hairpin to the terminal repeat region (Walther and Kennell 1999; Simpson et al. 2004). The full-length transcript potentially serves as both an mRNA for the reverse transcriptase and as a template for (-) strand cDNA synthesis. Following translation, the pFOXC-RT may bind directly to the RNA in cis and initiate cDNA synthesis at or near the extreme 3' end. Although the nature of the primer has yet to be determined, in vitro studies indicate that the pFOXC-RT can use DNA or snapped-back RNA primers. Due to the phylogenetic relationship with the pMaur-RT, the pFOXC-RT may also be able to initiate cDNA synthesis de novo. In addition, since the mature plasmid contains a 5'-linked protein, it is also conceivable that a protein is used as a primer. In this scenario, the RT may function as a primer itself, as has been shown for hepadnaviruses, such as the hepatitis B virus (reviewed in Ganem and Schneider 2001). Figure 5a shows four potential initiation mechanisms.

Characterization of the in vivo replication intermediates suggests that the telomere-like repeats may be added during reverse transcription (Simpson et al. 2004). A potential mechanism by which repeats may be added is illustrated in Fig. 5b, and is based on mechanisms used by protein-primed linear DNA elements (Salas, 1991). In this model, cDNA synthesis initiates via the extension of a snapped-back RNA and the pFOXC-RT pauses after copying a few nucleotides. The nascent cDNA-RT complex may slide back and become repositioned on the plasmid RNA, prior to elongation. This may occur more

Fig. 4 Hypothetical replication cycle of linear clothespin retroplasmids. Replication begins with transcription of the plasmid DNA by the host mt RNA polymerase. Transcription begins on the top strand and proceeds around the hairpin to the 3' repeat region (vertical bars). Transcripts (dotted line) appear to contain fewer 3' repeats than are associated with plasmid DNAs (three repeats vs. four repeats). The transcript serves as both an mRNA and a template for (-) strand cDNA synthesis. Following translation, the RT (diagonally-striped star) associates with the 3' end of the plasmid transcript where it initiates cDNA synthesis and may generate additional copies of the 5 bp repeat. Potential mechanisms used for initiation and repeat addition are shown in Fig. 5. Reverse transcription continues until the RT reaches the 5' end of the RNA template, generating a full-length (-) strand cDNA (dashed line). The 3' end of the (-) strand cDNA may serve as a primer for (+) strand synthesis, which is catalyzed by the plasmid RT or host DNA polymerase. The RNA template is degraded by a host RNAse H or displaced during the synthesis of the (+) strand. Following second strand synthesis, the RT (or mt DNA polymerase) may associate with the 5' end to potentially function as a protective cap

Fig. 4 Hypothetical replication cycle of linear clothespin retroplasmids. Replication begins with transcription of the plasmid DNA by the host mt RNA polymerase. Transcription begins on the top strand and proceeds around the hairpin to the 3' repeat region (vertical bars). Transcripts (dotted line) appear to contain fewer 3' repeats than are associated with plasmid DNAs (three repeats vs. four repeats). The transcript serves as both an mRNA and a template for (-) strand cDNA synthesis. Following translation, the RT (diagonally-striped star) associates with the 3' end of the plasmid transcript where it initiates cDNA synthesis and may generate additional copies of the 5 bp repeat. Potential mechanisms used for initiation and repeat addition are shown in Fig. 5. Reverse transcription continues until the RT reaches the 5' end of the RNA template, generating a full-length (-) strand cDNA (dashed line). The 3' end of the (-) strand cDNA may serve as a primer for (+) strand synthesis, which is catalyzed by the plasmid RT or host DNA polymerase. The RNA template is degraded by a host RNAse H or displaced during the synthesis of the (+) strand. Following second strand synthesis, the RT (or mt DNA polymerase) may associate with the 5' end to potentially function as a protective cap than once and would generate (-) strand cDNAs that have a greater number of repeats than associated with the RNA template.

Following the synthesis of a full-length (-) strand cDNA, the RNA template is presumably degraded by a host RNAse H, or displaced during second

Telomere Repeat Plasmid

Fig. 5 Models of initiation of cDNA synthesis and repeat addition. a Potential mechanisms used to initiate (-) strand cDNA synthesis of pFOXC3. The pFOXC3 transcript may snap back upon itself and the 3' hydroxyl of the terminal nucleotide is used as a primer (RNA snapback). A hydroxyl group may also be provided by a serine, threonine or tyrosine residue of the pFOXC-RT itself (protein priming) or from the 3' end of a cDNA or DNA molecule (DNA primer). Due to the phylogenetic relationship of pFOXC-RT to the pMaur-RT, cDNA synthesis may potentially initiate without a primer (de novo). b Addition of the telomere-like repeats. Following models for 3'-terminal repeat addition of linear DNAs that initiate via a protein primer (Salas 1991), the nascent pFOXC3 cDNA may slideback and reposition itself on the RNA template prior to being extended. As shown here, the pFOXC3-RT extends a snapped-back RNA and synthesizes a nascent cDNA of three nu-cleotides. The cDNA is displaced from the transcript and repositioned on the template prior to a new round of synthesis. This slideback-extension mechanism may occur more than once, generating multiple copies of the pentameric repeat, prior to elongation of the (-) strand cDNA (dashed line)

Fig. 5 Models of initiation of cDNA synthesis and repeat addition. a Potential mechanisms used to initiate (-) strand cDNA synthesis of pFOXC3. The pFOXC3 transcript may snap back upon itself and the 3' hydroxyl of the terminal nucleotide is used as a primer (RNA snapback). A hydroxyl group may also be provided by a serine, threonine or tyrosine residue of the pFOXC-RT itself (protein priming) or from the 3' end of a cDNA or DNA molecule (DNA primer). Due to the phylogenetic relationship of pFOXC-RT to the pMaur-RT, cDNA synthesis may potentially initiate without a primer (de novo). b Addition of the telomere-like repeats. Following models for 3'-terminal repeat addition of linear DNAs that initiate via a protein primer (Salas 1991), the nascent pFOXC3 cDNA may slideback and reposition itself on the RNA template prior to being extended. As shown here, the pFOXC3-RT extends a snapped-back RNA and synthesizes a nascent cDNA of three nu-cleotides. The cDNA is displaced from the transcript and repositioned on the template prior to a new round of synthesis. This slideback-extension mechanism may occur more than once, generating multiple copies of the pentameric repeat, prior to elongation of the (-) strand cDNA (dashed line)

strand synthesis. Due to the ability of the pFOXC-RT to copy DNA templates, the second strand may be synthesized by the pFOXC-RT; however, it is also possible that a host DNA polymerase is utilized. Once DNA synthesis is complete, the RT may dissociate from the plasmid DNA, or it may associate with the 5' end to potentially function as a protective cap.

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