Characterization of the C-terminal DNA-binding/DNA endonuclease region of a group II intron-encoded protein.
Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H-N-H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg(2+) ion at the H-N-H active site. Although the conserved cysteine residue pairs could potentially bind Zn(2+), the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn(2+), and the addition of exogenous Zn(2+) inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain.
San Filippo
Joseph
J
Department of Chemistry and Biochemistry, and Section of Molecular, Genetics and Microbiology, School of Biological Sciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, MBB2. 234BA, 2500 Speedway, Austin, TX 78712, USA.
Lambowitz
Alan M
AM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D003087:Colicins / Q000737:chemistry; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007438:Introns / Q000235:genetics*; D013294:Lactococcus lactis / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015032:Zinc / Q000378:metabolism
D001426:Bacterial Proteins; D002413:Cations, Divalent; D003087:Colicins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C099785:LtrA protein, Lactococcus lactis; D012194:RNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; C035571:endodeoxyribonuclease VII; D008274:Magnesium; D015032:Zinc