title: A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity.
abstract: L1 is a ubiquitous interspersed repeated sequence in mammals that achieved its high copy number by autonomous retrotransposition. Individual L1 elements within a genome differ in sequence and retrotransposition activity. Retrotransposition requires two L1-encoded proteins, ORF1p and ORF2p. Chimeric elements were used to map a 15-fold difference in retrotransposition efficiency between two L1 variants from the mouse genome, T(FC) and T(Fspa), to a single amino acid substitution in ORF1p, D159H. The steady-state levels of L1 RNA and protein do not differ significantly between these two elements, yet new insertions are detected earlier and at higher frequency in T(FC), indicating that it converts expressed L1 intermediates more effectively into new insertions. The two ORF1 proteins were purified and their nucleic acid binding and chaperone activities were examined in vitro. Although the RNA and DNA oligonucleotide binding affinities of these two ORF1 proteins were largely indistinguishable, D159 was significantly more effective as a nucleic acid chaperone than H159. These findings support a requirement for ORF1p nucleic acid chaperone activity at a late step during L1 retrotransposition, extend the region of ORF1p that is known to be critical for its functional interactions with nucleic acids, and enhance understanding of nucleic acid chaperone activity.
authors.affiliation: Department of Cell and Developmental Biology, University of Colorado School of Medicine, Aurora, CO 80045, USA. sandy.martin@ucdenver.edu
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Martin
authors.forename: Diane
authors.initials: D
authors.lastname: Bushman
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.forename: Patrick Wai-Lun
authors.initials: PW
authors.lastname: Li
authors.forename: Ann
authors.initials: A
authors.lastname: Walker
authors.forename: Jessica
authors.initials: J
authors.lastname: Cummiskey
authors.forename: Dan
authors.initials: D
authors.lastname: Branciforte
authors.forename: Mark C
authors.initials: MC
authors.lastname: Williams
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D020084:Long Interspersed Nucleotide Elements; D051379:Mice; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; C494023:L1 ORF1 protein, human; D018832:Molecular Chaperones; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D012313:RNA; D004247:DNA
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