Non-random features of loop-size chromatin fragmentation.
Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3'- to -5'-exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5' protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the approximately 50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements.
Szilágyi
Ildikó
I
Department of Biophysics and Cell Biology, University Medical School of Debrecen, 4012 Debrecen, Nagyerdei krt. 98, Hungary.
Varga
Tamás
T
Székvölgyi
Lóránt
L
Hegedüs
Eva
E
Goda
Katalin
K
Kaczur
Viktória
V
Bacsó
Zsolt
Z
Nakayama
Yuji
Y
Pósafi
János
J
Pongor
Sándor
S
Szabó
Gábor
G
D000818:Animals; D001483:Base Sequence; D002843:Chromatin / Q000737:chemistry* / Q000302:isolation & purification*; D053938:DNA Fragmentation; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I; D015180:Electrophoresis, Gel, Two-Dimensional; D018922:HL-60 Cells; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D041681:NIH 3T3 Cells; D017422:Sequence Analysis, DNA / Q000379:methods
D002843:Chromatin; D004253:DNA Nucleotidylexotransferase; D004256:DNA Polymerase I