Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA. Evidence for both major and minor groove contacts.
Beta-D-Glucosyl-hydroxymethyluracil, also called base J, is an unusually modified DNA base conserved among Kinetoplastida. Base J is found predominantly in repetitive DNA and correlates with epigenetic silencing of telomeric variant surface glycoprotein genes. We have previously identified a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia, and we have shown that it is a structure-specific binding protein. Here we examine the molecular interactions that contribute to recognition of the glycosylated base in synthetic DNA substrates using modification interference, modification protection, DNA footprinting, and photocross-linking techniques. We find that the two primary requirements for J-DNA recognition include contacts at base J and a base immediately 5' of J (J-1). Methylation interference analysis indicates that the requirement of the base at position J-1 is due to a major groove contact independent of the sequence. DNA footprinting of the JBP.J-DNA complex with 1,10-phenanthroline-copper demonstrates that JBP contacts the minor groove at base J. Substitution of the thymine moiety of J with cytosine reduces the affinity for JBP approximately 15-fold. These data indicate that the sole sequence dependence for JBP binding may lie in the thymine moiety of base J and that recognition requires only two specific base contacts, base J and J-1, within both the major and minor groove of the J-DNA duplex.
Sabatini
Robert
R
Division of Geographic Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. sabatini@uab.edu
Meeuwenoord
Nico
N
van Boom
Jacques H
JH
Borst
Piet
P
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003421:Crithidia; D019175:DNA Methylation; D016054:DNA, Protozoan / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005960:Glucosides; D007891:Leishmania; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D015800:Protozoan Proteins / Q000378:metabolism; D014345:Trypanosoma; D014498:Uracil / Q000031:analogs & derivatives*
D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D005960:Glucosides; C402790:J-specific DNA-binding protein, protozoa; D015800:Protozoan Proteins; C084880:5-((glucopyranosyloxy)methyl)uracil; D014498:Uracil