DNA polymerization by the reverse transcriptase of the human L1 retrotransposon on its own template in vitro.
L1 elements (LINE-1s) account for 17% of the human genome and have achieved this abundance by transpositions via an RNA intermediate, or retrotransposition. Reverse transcription is a crucial event in the retrotransposition of the active human L1 element and is carried out by the L1-encoded ORF2 protein. Previously, we performed biochemical characterization of the human L1 ORF2 protein with reverse transcriptase (RT) activity (referred to as L1 RT), expressed in baculovirus-infected insect cells. In the present study, we describe the properties of DNA- and RNA-dependent DNA synthesis catalyzed by the L1 RT on the L1 templates in vitro. We found that L1 RT synthesized at least 620 of nucleotides per template binding event utilizing L1 RNA in vitro. Under processive conditions the L1 RT synthesized cDNA over 5 times longer than that Moloney murine leukemia virus RT on the heteropolymeric RNA template used in these studies. These data are the first to demonstrate that RT from the human L1 element is a highly processive polymerase among RT enzymes. This report also presents a strong evidence of lack of RNase H activity for the L1 ORF2 protein in vitro, distinguishing L1 RT from retroviral RTs. Finally, we found strong pausing for of the L1 RT during DNA polymerization within the 3' untranslated region of L1 mRNA, that is result of contribution both rGs runs of the polypurine stretch and immediately adjacent stem-loop structure. A mechanism facilitating minus-strand DNA synthesis during reverse transcription of L1 element in vivo is discussed.
Piskareva
Olga
O
Institute of Biochemistry and Physiology of Microorganisms RAS Pushchino, Prosoekt Nauki 5, 142290 Pushchino, Moscow region, Russia.
Schmatchenko
Vadim
V
D000818:Animals; D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000502:physiology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000201:enzymology / Q000235:genetics; D009690:Nucleic Acid Conformation; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics*; D016914:Ribonuclease H / Q000378:metabolism
D036022:Capsid Proteins; D006023:Glycoproteins; D018626:Retroelements; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H