Replicative form circularization

The genomes of DNA viruses vary greatly in size (Table 24-1). Many viral DNAs are circular for at least part of their life cycle. During viral replication within a host cell, specific types of viral DNA called replicative forms may appear for example, many linear DNAs become circular and all single-stranded DNAs become... 

When the "replicative form" of DNA of the virus (()X174 (Chapter 27), a small circular molecule containing 5000 bp, was treated with proflavine, the binding of 0.06 mol of proflavine per mole of nucleotides reduced Wr to zero. From this it was estimated that o = 0.055, corresponding to -27 superhelical turns at 25°, pH 6.8, ionic strength 0.2. 

Goulian, M. and Kornberg, A. (1%7) Enzymatic synthesis of DNA. 23. Synthesis of circular replicative form of... 

Digested phagemid DNA vector should run as a 4.5 kb linear double-stranded fragment. If there is any residual uncut closed circular replicative form (which runs at the position of a 2.5 kb supercoiled DNA), digjest further with Sfil. 

Suitable vectors have been derived from the bacteriophage M13 which, except in its replicating form (RF), contains single-stranded circular DNA. The derivative M13 mpl, produced by insertion of part of the Escherichia coli lac operon, was mutated to give a single site for the restriction endo nuclease EcoRl within the gene for /8-D-galactosidase. This enzyme produces a blue colour when the hosts of this... 

Chandler, B., Hayashi, M., Hayashi, M.N., and Spiegeltrtan, S. (1964) Circularity of the replicating form of a single-stranded DNA vims. Science, 143,47 9. 

It thus appears that many, if not all DNA s of viral or bacterial origin, even if linear In the vegetative form, circularize during replication and that the repetitive end sequences serve as locks to fuse the circles. Most of the closed circular DNA s have an additional feature they are super-coiled, i.e., they lack a certain constant number of helix turns and will therefore compensate for this strain by super-coiling. They are thus packed more tightly. The supercoil structure of circular DNA s can be relaxed spontaneously by single-stranded breaks or gradually (Fig. 5.10) by Intercalation of such aromatic dyes as ethidlum bromide (32) (see Chapter 7). 

The circular molecule of single-stranded DNA of phage ( X174 does not lose its circular structure during cell auto replication when s5Tithesizing its own complementary copy and forming the double-helical replicative form. The newly formed replicative form also has the structure of a closed ring, as has been shown not only by indirect biochemical methods (Burton and Sinsheimer,... 

As the results of molecular hybridization and determination of base sequence have shown, RNA formed during phage reproduction in vivo on the basis of the circular double-helical replicative form of DNA (Hayashi et al., 1964) is complementary to only one of the two DNA chains, i.e., to that which is complementary, in turn, to the single-stranded DNA of the mature phage. In vitro when the circular structure of DNA is disturbed, both strands of replicative DNA can act as substrate for RNA-polymerase. The single-stranded DNA of this phage can also act as template for RNA synthesis in vitro, with the preliminary formation of DNA/ RNA hybrids (Chamberlin and Berg, 1964). 

Conjugative transposons are self-transmissible large DNA elements (up to 150kbp) located in the donor chromosome. After excision a circular intermediate is formed that is unable to replicate autonomously. It is nicked at a origin of transfer site and one strand is then transferred to the recipient cell. After generating a double-stranded circle the transposon integrates into the recipients chromosome. 

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