Recognition of base J in duplex DNA by J-binding protein.
beta-d-Glucosylhydroxymethyluracil, also called base J, is an unusual 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 found a J-binding protein (JBP) in Trypanosoma, Leishmania, and Crithidia. We have now characterized the binding properties of recombinant JBP from Crithidia using synthetic J-DNA substrates that contain the glycosylated base in various DNA sequences. We find that JBP recognizes base J only when presented in double-stranded DNA but not in single-stranded DNA or in an RNA:DNA duplex. It also fails to interact with free glucose or free base J. JBP is unable to recognize nonmodified DNA or intermediates of J synthesis, suggesting that JBP is not directly involved in J biosynthesis. JBP binds J-DNA with high affinity (K(d) = 40-140 nm) but requires at least 5 bp flanking the glycosylated base for optimal binding. The nature of the flanking sequence affects binding because J in a telomeric sequence binds JBP with higher affinity than J in another sequence known to contain J in trypanosome DNA. We conclude that JBP is a structure-specific DNA-binding protein. The significance of these results in relation to the biological role and mechanism of action of J modification in kinetoplastids is discussed.
Sabatini
Robert
R
Division of Molecular Biology and Centre for Biomedical Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. sabatini@uab.edu
Meeuwenoord
Nico
N
van Boom
Jacques H
JH
Borst
Piet
P
D000818:Animals; D016831:Crithidia fasciculata / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D015394:Molecular Structure; D009841:Oligonucleotides / Q000378:metabolism; D011485:Protein Binding; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014498:Uracil / Q000031:analogs & derivatives* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D005960:Glucosides; C402790:J-specific DNA-binding protein, protozoa; D009841:Oligonucleotides; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C084880:5-((glucopyranosyloxy)methyl)uracil; D014498:Uracil; D004247:DNA