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Quantitative analysis of distribution of chromosomal proteins on single copy DNA sequences has been further developed. Our approach consists of DNAprotein crosslinking within whole cells or isolated nuclei, specific Immunoaffinity isolation of crosslinked complexes via protein and Identification of crosslinked DNA by hybridisation with single-stranded DNA probes. The present study shows that transcribed chromatin of chicken embryonic erythrocyte β globin gene is characterized by about 1.5 – 2.5-fold higher density of HMG 14/17 and 2-fold lower density of H1 and H5 as compared with non-transcribed chromatin of ovalbumin and lysozyrne genes, whereas HMG 1/2,E proteins were equally distributed between DNA of both transcribed and non-transcribed genes. The depletlon of H1/H5 in β globin sequences was verified by the ‘protein image’ hybridisation technique (1). The DNase I hypersensitive site located 5′ upstream from β globin gene is deficient in all the proteins assayed, what implies a drastic disruption in the nucleosomal array. Minor quantttative changes of protein pattern suggest transient local perturbation of the chromatin on transcription.

A study was made of the interaction between restriction endonucleases recognizing CCNGG (Ssoll and ScrFl) or CCA/TGG (Mval and EcoRll) DNA sequences and a set of synthetic substrates containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1′-hydroxy-2′-(hydroxymethyl)ethoxy] methylguanine (glG) residues replacing either one of the central nucleosides or dG residues in the recognition site. The non-nucleotide inserts (except for glG) introduced into the recognition site both increase the efficiency of Ssoll and change its specificity. A cleavage at the noncanonical position takes place, in some cases in addition to the correct ones. Noncanonical hydrolysis by Ssoll occurs at the phosphodiester bond adjacent to the point of modification towards the 5′-end. With the guanine base returned (the substrate with glG), the correct cleavage position is restored. ScrFl specifically cleaves all the modified substrates. DNA duplexes with non-nucleotide inserts (except for the glG-containing duplex) are resistant to hydrolysis by Mval and EcoRll. Prompted by the data obtained we discuss the peculiarities of recognition by restriction endonucleases of 5-membered DNA sequences which have completely or partially degenerated central base pairs. It is suggested that Ssoll forms a complex with DNA in an ‘open’ form.

The regulation of the SsoII restriction-modification system from Shigella sonnei was studied in vivo and in vitro. In lacZ fusion experiments, SsoII methyltransferase (M.SsoII) was found to repress its own synthesis but stimulate expression of the cognate restriction endonuclease (ENase). The N-terminal 72 amino acids of M.SsoII, predicted to form a helix-turn-helix (HTH) motif, was found to be responsible for the specific DNA-binding and regulatory function of M.SsoII. Similar HTH motifs are predicted in the N-terminus of a number of 5-methylcytosine methyltransferases, particularly M.EcoRII, M.dcm and M.MspI, of which the ability to regulate autogenously has been proposed. In vitro, the binding of M.SsoII to its target DNA was investigated using a mobility shift assay. M.SsoII forms a specific and stable complex with a 140 bp DNA fragment containing the promoter region of SsoII R-M system. The dissociation constant (Kd) was determined to be 1.5 × 10−8 M. DNaseI footprinting experiments demonstrated that M.SsoII protects a 48–52 bp region immediately upstream of the M.SsoII coding sequence which includes the predicted −10 promoter sequence of M.SsoII and the −10 and −35 sequences of R.SsoII.

Oligonucleotldes containing 2-amlnopurine (2-AP) In place of G or A in the recognition site of EcoRII (CCT/AGG) or Ssoll (CCNGG) restriction endonucleases have been synthesized In order to Investigate the specific interaction of DNA with these enzymes. Physicochemlcal properties (CD spectra and melting behaviour) have shown that DNA duplexes containing 2-aminopurine exist largely in a stable B-IIke form. 2-Aminopurine base paired with cytidine, however, essentially Influences the helix structure. The presence of a 2-APC mismatch strongly reduces the stability of the duplexes in comparison with the natural double strand, indicated by a blphaslc melting behaviour. Ssoil restriction endonuclease recognizes and cleaves the modified substrate with a 2-APT mismatch In the centre of the recognition site, but it does not cleave the duplexes containing 2-amlnopurine in place of inner and outer G, or both. EcoRII restriction endonuclease does not cleave duplexes containing 2-amlnopurine at all. The two-substrate mechanism of EcoRII-DNA interaction, however, allows hydrolysis of the duplex containing 2-amlnopurine In place of adenine In the presence of the canonical substrate.

Two different cytosine DHA-methylases, NI and GII, are present in Escherichia coli SK. The GII methylase recognizes the five-member symmetric sequence: 5′…NpCpCpApGpGpN…3′. This sequence is identical with the recognition site of the hsp II type determined by RII plasmide but, in contrast to RII methylase, the GII enzyme methylates cytosine located on the 5′ side of the site. By analogy with the isoshizomery of the restricting endonucleases, RII and GII DNA methylaeses may be called isomethymers which recognize the same site but methylate different bases. Since the phage of the SK and hsp II phenotypes is effectively restricted in respective cells it may be assumed that the isomethymeric modification does not provide any protection against the corresponding restrictases. NI methylase recognizes the five-member symmetric site which represents an inverted sequence of the GTT site: 5′…HpGpGpApCpCpN…3′. In this ease cytosine at the 3′-end of the recognition site is methylated.