Origin of DNA replication in papovavirus chromatin is recognized by endogenous endonuclease.

A portion of the nucleoprotein containing viral DNA extracted from cells infected by simian virus (SV40) is preferentially cleaved by endonucleases in a region of the genome encompassing the origin of replication and early and late promoters. To explore this nuclease-sensitive structure, we cleaved SV40 chromatin molecules with restriction enzymes and digested the exposed termini with nuclease Bal3l. Digestion proceeded only a short distance in the late direction from the MspI site, but some molecules were degraded 400 to 500 base pairs in the early direction. By comparison, BglI-cleaved chromatin was digested for only a short distance in the early direction, but some molecules were degraded 400 to 450 base pairs in the late direction. These barriers to Bal3l digestion (bracketing the BglI and the MspI sites) define the borders of the same open region in SV40 chromatin that is preferentially digested by DNase I and other endonucleases. In a portion of the SV40 chromatin, Bal3l could not digest through the nuclease-sensitive region and reached barriers after digesting only 50 to 100 base pairs from one end or the other. Chromatin molecules that contain barriers in the BglI to MspI region are physically distinct from molecules that are open in this region as evidenced by partial separation of the two populations on sucrose density gradients.Viral nucleoprotein complex extracted from cells infected by simian virus 40 (SV40) resembles cellular chromatin (1,10,15,27). A short segment of the viral genome (ca. 400 base pairs [bp] extending from the SV40 origin of replication in the late direction) is hypersensitive to endonuclease cleavage (33,(38)(39)(40) and appears, in the electron microscope, as a region which is free of nucleosomes (19,32). Recently, more detailed studies have shown that, within the nuclease-sensitive site, there are subregions with differential sensitivity to DNase 1 (7,8,31,32). Presumably, these features reflect the structural details of the nucleoprotein within this region.Nuclease-hypersensitive sites usually occur over regions which contain genetic signals. Such a chromatin structure over a eucaryotic promoter may be a necessary (but not sufficient) condition for transcription from that promoter (6,12,43). In addition, hypersensitive sites have been reported which are probably not related to promoter function (3,22,28). SV40-infected cells provide a good opportunity to investigate the details of the nuclease-sensitive chromatin structure, since SV40 chromatin is present in high concentration by comparison with other genes and can easily be separated from bulk cellular chromatin.We have developed a new approach for probing nucleoprotein structure which should be applicable to the study of chromatin structure in other minichromosomes. SV40 is cleaved with a restriction enzyme which cuts at a unique position, and the exposed termini are digested with Bal3l, an exonuclease which can degrade from either 3' or 5' ends. MATERIALS AND METHODSGrowth of cells and virus infections. African green monk...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Barriers to nuclease Bal31 digestion across specific sites in simian virus 40 chromatin.

Scott¹,

Walter²,

Cryer³

1984

“…The availability of an amplified, homogeneous DNA species in a chromatin-like structure has provided convenient material for the study of specific features of chromatin. Of particular interest is the short region adjacent to the viral origin of replication (ori site) which is hypersensitive to cleavage by endonucleases (23,26,27,29) and which appears as a gap in the nucleosome pattern when visualized in the electron microscope (16,22). This region contains the early and late gene promoters, the transcriptional enhancer sequences, and the viral origin of replication.…”

mentioning

confidence: 99%

Role of specific simian virus 40 sequences in the nuclease-sensitive structure in viral chromatin.

Gerard¹,

Montelone²,

Walter³

et al. 1985

A nuclease-sensitive region forms in chromatin containing a 273-base-pair (bp) segment of simian virus 40 DNA encompassing the viral origin of replication and early and late promoters. We have saturated this region with short deletion mutations and compared the nuclease sensitivity of each mutated segment to that of an unaltered segment elsewhere in the partially duplicated mutant. Although no single DNA segment is required for the formation of a nuclease-sensitive region, a deletion mutation (d145) which disrupted both exact copies of the 21-bp repeats substantially reduced nuclease sensitivity. Deletion mutations limited to only one copy of the 21-bp repeats had little, if any, effect. A mutant (d1135) lacking all copies of the 21-and 72-bp repeats, while retaining the origin of replication and the TATA box, did not exhibit a nuclease-sensitive region. Mutants which showed reduced nuclease sensitivity had this effect throughout the nuclease-sensitive region, not just at the site of the deletion, indicating that although multiple determinants must be responsible for the nuclease-sensitive chromatin structure they do not function with complete independence. Mutant d19, which lacks the late portion of the 72-bp segment, showed reduced accessibility to BglI, even though the Bgll site is 146 bp away from the site of the deletion.Simian virus 40 (SV40)-infected cells contain SV40 DNA in a nucleoprotein structure with many similarities to cellular chromatin. The availability of an amplified, homogeneous DNA species in a chromatin-like structure has provided convenient material for the study of specific features of chromatin. Of particular interest is the short region adjacent to the viral origin of replication (ori site) which is hypersensitive to cleavage by endonucleases (23,26,27,29) and which appears as a gap in the nucleosome pattern when visualized in the electron microscope (16,22). This region contains the early and late gene promoters, the transcriptional enhancer sequences, and the viral origin of replication. The distinctive chromatin structure may be relevant to any or all of these functions.To identify which sequences are responsible for the nuclease-sensitive region, partially duplicated SV40 mutants have been studied. All essential information required for the hypersensitive site is contained within a 273-base-pair (bp) segment of DNA spanning the origin of replication (11,30 We have investigated the genetic elements responsible for the chromatin structure in this region by analyzing a number of additional mutants. No single segment is essential for nuclease sensitivity. However, overall accessibility to nuclease was dramatically decreased by a mutant affecting only the two exact copies of the 21-bp repeated sequences. Insertion of a foreign DNA segment into this region also substantially diminished nuclease sensitivity. By contrast, deletion of only one of the two exact copies of the 21-bp repeats had little effect on the pattern of nuclease cleavage.These results imply that the region of the genome e...

“…Data reported in the literature suggest the occurrence of nucleosome phasing at the SV40 regulatory region (15,16,23,24), although the molecular basis of this process is obscure. Our results provide the first experimental evidence suggesting that there is a preferred phasing of the histone octamers within the ori region of SV40.…”

Section: Discussionmentioning

confidence: 99%

Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nobile¹,

Nickol²,

Martin³

1986

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.Palindromic (inverted repeat) DNA sequences are of biological interest because they are often located in functionally important regions of DNA and have the potential to form cruciform structures. Attempts to demonstrate the existence of cruciform structures in vivo have so far been unsuccessful (19). However, the possibility that cruciform structures exist at least transiently in vivo is suggested by the observations that long inverted repeats are generally unstable (8) and that biologic functions in some cases can be regained by second site mutations that restore the possibility of forming stemloop structures (reviewed in reference 1; L. RothmanDenes, personal communication; although these examples may be due to stem-loop structures of the RNA transcript [14]).Ordinarily, palindromic sequences do not form stable cruciform structures in solution unless the DNA is negatively supercoiled (3,4,8). Since supercoiled DNA is not suitable for experiments with very short DNA fragments, we constructed a set of simian virus 40 (SV40) mutants containing rearrangements at the 27-base-pair (bp) palindrome located at the viral origin of DNA replication and utilized them to generate cruciform (double stem-loop)-containing heteroduplex DNA molecules.We have reported elsewhere (C. Nobile and R. G. Martin, Int. Virol., in press) the construction and characterization of single and double stem-loop heteroduplexes. These molecules obviously do not require supercoiling to be stable in solution. Another heteroduplex, having a stable stem-loop structure, 7-bp stem, and 3-base loop on only one of the two strands, was previously utilized for in vitro nucleosome reconstitution (10). Restriction analyses of reconstituted nucleosomes indicated that the single stem-loop structure was preferentially located b...