Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed.
BACKGROUND
The pFOXC retroplasmids are small, autonomously replicating DNA molecules found in mitochondria of certain strains of the filamentous fungus Fusarium oxysporum and are among the first linear genetic elements shown to replicate via reverse transcription. The plasmids have a unique clothespin structure that includes a 5'-linked protein and telomere-like terminal repeats, with pFOXC2 and pFOXC3 having iterative copies of a 5 bp sequence. The plasmids contain a single large open reading frame (ORF) encoding an active reverse transcriptase (RT). The pFOXC-RT is associated with the plasmid transcript in a ribonucleoprotein (RNP) complex and can synthesize full-length (-) strand cDNA products. In reactions containing partially purified RT preparations with exogenous RNAs, the pFOXC3-RT has been shown to initiate cDNA synthesis by use of snapped-back RNAs, as well as loosely associated DNA primers.


RESULTS
The complete sequence of the distantly related pFOXC1 plasmid was determined and found to terminate in 3-5 copies of a 3 bp sequence. Unexpectedly, the majority of (-) strand cDNA molecules produced from endogenous pFOXC1 transcripts were attached to protein. In vitro experiments using partially purified pFOXC3-RT preparations having a single radiolabeled deoxyribonucleotide triphosphate (dNTP) generated a nucleotide-labeled protein that migrated at the size of the pFOXC-RT. The nucleotide preference of deoxynucleotidylation differed between pFOXC3 and pFOXC1 and showed complementarity to the respective 3' terminal repeats. In reactions that include exogenous RNA templates corresponding to the 3' end of pFOXC1, a protein-linked cDNA product was generated following deoxynucleotidylation, suggesting that reverse transcription initiates with a protein primer.


CONCLUSIONS
The finding that reverse transcription is protein primed suggests the pFOXC retroplasmids may have an evolutionary relationship with hepadnaviruses, the only other retroelement family known to initiate reverse transcription via a protein primer. Moreover, the similarity to protein-primed linear DNA elements supports models in which the terminal repeats are generated and maintained by a DNA slideback mechanism. The ability of the pFOXC-RT to utilize RNA, DNA and protein primers is unique among polymerases and suggests that the pFOXC plasmids may be evolutionary precursors of a broad range of retroelements, including hepadnaviruses, non-long terminal repeat (non-LTR) retrotransposons and telomerase.
Department of Biology, Saint Louis University, St Louis, MO, USA. kennellj@slu.edu.
Jeffrey T
JT
Galligan
Sarah E
SE
Marchetti
John C
JC
Kennell
Cell. 2002 Nov 1;111(3):433-44
12419252
Mol Cell Biol. 1992 Nov;12(11):5131-44
1383691
Methods Enzymol. 1979;59:421-33
440084
Biol Bull. 1999 Jun;196(3):327-8; discussion 329-30
10390830
Eukaryot Cell. 2004 Dec;3(6):1589-600
15590832
J Virol. 1993 Aug;67(8):4513-20
7687299
Appl Environ Microbiol. 1997 Aug;63(8):3311-3
9251222
Science. 1997 Aug 15;277(5328):911-2
9281073
J Bacteriol. 1986 Aug;167(2):587-93
3015880
Cell. 1986 Aug 29;46(5):669-80
2427199
Cell. 1992 Nov 13;71(4):663-70
1384989
Cell. 1993 Apr 9;73(1):133-46
7681727
J Virol. 1994 May;68(5):2994-9
7512155
Mol Cell. 1999 Aug;4(2):229-38
10488338
J Virol. 1994 Jan;68(1):6-13
7504742
Mol Cell Biol. 1981 Feb;1(2):165-78
6100962
Cell. 1993 Dec 17;75(6):1071-81
7505202
Nucleic Acids Res. 1993 Feb 11;21(3):641-7
8441675
J Biol Chem. 2003 Nov 7;278(45):43951-60
12937178
Curr Top Microbiol Immunol. 2003;272:187-211
12747551
Cytogenet Genome Res. 2005;110(1-4):475-90
16093701
Curr Genet. 2000 Dec;38(5):283-90
11191213