Deoxyribonuclease II as a probe to sequence-specific chromatin organization: preferential cleavage in the 72 bp modulator sequence of SV40 minichromosome

Abstract

T prove sequence-specific chromatin structure of SV40 minichromosome, we further modified previously described hybridization mapping. Actually (i) the digestion patterns by two nucleases (micrococcal and DNAase II) were compared and (ii) the kinetics of nuclease digestion was analyzed from early time points when only a fraction of minichromosomes was cleaved once to longer digestions when oligo- and mononucleosomal bands appeared. DNAase II is shown to possess certain sequence specificity different from that of micrococcal nuclease. The major finding is that DNAase II preferentially cleaves the SV40 minichromosome at a distinct region of the genome known as 72 bp modulator element. Other hypersensitive sites are located near the replication origin and T-ag binding site II and also near BamHI site where termination of replication and "late" transcription occurs. Micrococcal nuclease splits the BglI-Hpaii region in a different manner.

Identification within the simian virus 40 genome of a chromosomal loop attachment site that contains topoisomerase II cleavage sites

“…It has been demonstrated that this region is preferentially sensitive to Si nuclease (2), prefers to bind gene 32 protein of bacteriophage T4 (33), reacts preferentially with carbodiimides (7), and is hypersensitive to micrococcal nuclease (Pommier et al, unpublished results). In addition, minor DNase I and DNase II hypersensitive sites reside at or near this region in SV40 minichromosomes (13,42). Whether the SV40 MAR has a cis-acting effect on the biological functions of the virus is a key question remaining to be answered, particularly since in studies to be reported elsewhere, the K gene MAR has been shown to exert a positive quantitative effect on gene expression (Blasquez et al and Xu et al, in press).…”

Control Region of SV40 Minichromosomes is Preferentially Cleaved by Single-Strand Specific SI Nuclease

“…Extraction and purification of the minichromosomes were performed as described previously (13) with minor modifications. Extraction buffer contained 0.1 M NaCl, 10 mM triethanolamine-HCl, pH 7.8, 0.1 mM EDTA, 0.5 mM PMSF, 0.5% NP40.…”