N. Ramesh

Recognition of B and Z forms of DNA by Escherichia coli DNA polymerase I

Since the substrate binding domain of the large proteolytic fragment of Escherichia coli DNA polymerase I has been shown to interact with the B forms of DNA, we have studied the ability of this enzyme to recognize structures other than the B form. The polymerase activity has been used to evaluate the degree of recognition of the B and Z forms of DNA. The Z form was found to promote less activity, indicating the probable inability of the polymerase to move along the conformationally rigid form of the template. The present study indicates that the Z-DNA found in vivo may have a role in the control of replication.

Critical cation balance in B → Z transition: role of Li+

The sodium salt of poly(dG‐dC) is known to exhibit a B → Z transition in the presence of various cations and 60% alcohol. We here show that the lithium salt of poly(dG‐dC) does not undergo B → Z transition in the presence of 60% alcohol since Li+ with its large hydration shell cannot stabilize the Z‐form. On the other hand, high concentrations of Mg2+ or micromolar concentrations of the cobalt hexamine complex which are known to stabilize the Z‐form can compete with Li+ for charge neutralization and hence bring about a B → Z transition in the same polymer. From the model building studies the mode of action of the cobalt—hexamine complex in stabilizing the Z‐form is postulated.

Zintrons in rat α-lactalbumin gene

The eukaryotic genome is characterised by the occurrence of a large amount of repetitive DNA which exists within and around coding sequences. Random repeats of (TG) n sequences have been observed in several introns. In this paper we show that (TG)14 and (TG)24 sequences present in the third intron of rat α‐lactalbumin gene adopt left‐handed Z‐helices under varying superhelical densities. The overall sequence of the parent plasmid further influenced the level of supercoiling at which the B to Z transition could be induced in (TG) n sequences. Such Z‐potential intervening sequences (zintrons) could act as buffers to maintain desired levels of supercoiling near the transcribed sequences.

Left handed DNA in synthetic and topologically constrained form V DNA and its implications in protein recognition

We have investigated structural transitions in Poly(dG-dC) and Poly(dG-Me5dC) in order to understand the exact role of cations in stabilizing left-handed helical structures in specific sequences andthe biological role, if any, of these structures. From a novel temperature dependent Z ⇌ B transition it has been shown that a minor fluctuation in Na+ concentration at ambient temperature can bring about B to Z transition. Forthe first time, wehave observed a novel Z⇌B⇌Zuble transition in poly(dG-Me5dC) as the Na+ concentration is gradually increased. This suggests that a minor fluctuation in Na+ concentration in conjunction with methylation may transform small stretches of CG sequences from one conformational state to another. These stretches could probably serve as sites for regulation. Supercoiled formV DNA reconstituted from pBR322 and pβG plasmids have been studied as model systems, in order to understand the nature and role of left-handed helical conformation in natural sequences. A large portion of DNA in form V, obtained by reannealing the two complementary singlestranded circles is forced to adopt left-handed double helical structure due to topological constraints (Lk = 0). Binding studies with Z-DNA specific antibody and spectroscopic studies confirm the presence of left-handed Z-structure in the pβG and pβR322 form V DNA. Cobalt hexamine chloride, which induces Z-form in Poly(dG-dC) stabilizes the Z-conformation in form V DNA even in the non-alternating purine-pyrimidine sequences. A reverse effect is observed with ethidium bromide. Interestingly, both topoisomerase I and II (from wheat germ) act effectively on form V DNA to give rise to a species having an electrophoretic mobility on agarose gel similar to that of open circular (form II) DNA. Whether this molecule is formed as a result of the left-handed helical segments of form V DNA undergoing a transition to the right-handed B-form during the topoisomerase action remains to be solved.

Role of the environment in the interaction of nonintercalators with Z-DNA.

Interaction of the DNA binding nonintercalators Netropsin, Distamycin and the mPD derivative with Z-DNA has been studied. It has been found that environmental factors like the solvent and added cations significantly modulate the interaction of these ligands with Z-DNA. However no definite Z to B transition in presence of these ligands was found in any case, in contrast to previously reported results (Ch. Zimmer, C. Marck and W. Guschlbauer, FEBS Lett. 154, 156-160 (1983)).

A new polymer supported packing material as a substitute of hydroxylapatite: Purification of Topoisomerases

SummaryHydroxylapatite (HA), a form of calcium phosphate, finds extensive usages in the fractionation and purification of proteins, enzymes and nucleic acids. However, commercial HA preparations for liquid chromatography (LC) differ from many other LC packing materials in that the former is not polymer supported. We have now developed a substitute packing material which uses Fractosil 200 as the polymer support. Other polymer supports can also be similarly used. Being Fractosil 200 supported, this Hydroxylapatite Substitute (HAS) matrix can be employed for open column LC as well as for HPLC purposes. Although the exact chemical nature of HAS is different from the conventional HA, HAS mimicks the functions of the latter support. Due to the high’ binding capacity of HA many DNA binding proteins have been partially purified on this support. In the present work HAS has been successfully used in the partial purification of Topoisomerase I and II from wheat germ.

Structural alteration from non-B to B-form could reflect DNase I hypersensitivity.

Preferential cleavage of active genes by DNase I has been correlated with a structurally altered conformation of DNA at the hypersensitive site in chromatin. To have a better understanding of the structural requirements for gene activation as probed by DNase I action, digestability by DNase I of synthetic polynucleotides having the ability to adopt B and non-B conformation (like Z-form) was studied which indicated a marked higher digestability of the B-form of DNA. Left handed Z form present within a natural sequence in supercoiled plasmid also showed marked resistance towards DNase I digestion. We show that alternating purine-pyrimidine sequences adopting Z-conformation exhibit DNAse I foot printing even in a protein free system. The logical deductions from the results indicate that 1) altered structure like Z-DNA is not a favourable substrate for DNase I, 2) both the ends of the alternating purine-pyrimidine insert showed hypersensitivity, 3) B-form with a minor groove of 12-13 A is a more favourable substrate for DNase I than an altered structure, 4) any structure of DNA deviating largely from B form with a capacity to flip over to the B-form are potential targets for the DNase I enzymic probes in naked DNA.

Screening of clones containing altered DNA conformations by plasmid topoprofile shift

DNA topoprofile of supercoiled plasmid is influenced by the presence of altered structures in some stretches. We describe a non-radioactive method for screening the clones containing such altered structures.

Implementation of an interactive relational graphics database

The benefits that accrue from the use of design database include (i) reduced costs of preparing data for application programs and of producing the final specification, and (ii) possibility of later usage of data stored in the database for other applications related to Computer Aided Engineering (CAE). An INTEractive Relational GRAphics Database (INTERGRAD) based on relational models has been developed to create, store, retrieve and update the data related to two dimensional drawings. INTERGRAD provides two languages, Picture Definition Language (PDL) and Picture Manipulation Language (PML). The software package has been implemented on a PDP 11/35 system under the RSX-11M version 3.1 operating system and uses the graphics facility consisting of a VT-11 graphics terminal, the DECgraphic 11 software and an input device, a lightpen.