Insights into the mechanism of DNA recognition by the methylated LINE binding protein EhMLBP of Entamoeba histolytica.
EhMLBP is an essential Entamoeba histolytica protein that binds preferentially to methylated long interspersed nuclear elements and rDNA. In an effort to identify more EhMLBP DNA substrates, we developed an affinity-based technique in which the C-terminal DNA binding domain of EhMLBP (GST-CterEhMLBP) was used as the ligand. Bioinformatic analysis of the DNA sequences that were isolated by this affinity method revealed the presence of a 29-nucleotide consensus motif that includes a stretch of ten adenines. Gel retardation analysis showed that EhMLBP binds to the consensus motif with a preference for its methylated form. Four DNA sequences, namely those that encoded either dihydrouridine synthetase, RAP GTPase activating protein, serine/threonine protein kinase or leucine-rich repeat containing protein (LRPP) were then selected for further analysis. In vivo binding of EhMLBP to these genes was confirmed by chromatin immunoprecipitation. The presence of methylated cytosines was detected in DNA encoding LRPP and to a lower extent in the other genes. EhMLBP binds preferentially to the methylated forms of these DNA targets. The ability of the consensus motif to compete with EhMLBP binding to its DNA substrates indicates that the adenine stretch is involved in the mechanism of DNA recognition. The results of this investigation extend our existing knowledge on the number of DNA sequences that are recognized by EhMLBP and reinforce the notion that this protein is an innate methylated DNA binding protein in E. histolytica.
Lavi
Tal
T
Department of Molecular Microbiology, Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Siman-Tov
Rama
R
Ankri
Serge
S
D020816:Amino Acid Motifs; D000818:Animals; D019175:DNA Methylation; D016054:DNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004748:Entamoeba histolytica / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020084:Long Interspersed Nucleotide Elements; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D015800:Protozoan Proteins