Gapped duplex

Mills JB, Vacano E, Hagerman PJ (1999) Flexibility of single-stranded DNA use of gapped duplex helices to determine the persistence lengths of poly(dT) and poly(dA). J Mol Biol 285 245-257... 

Gapped duplexes. The nicks introduced into single strands can be broadened into gaps by the action of exonuclease III (Fig. 1.3c.)... 

FIGURE 6.1 Template specificity of E. colt DNA Pol 1 and Pol Ik. (a) Typical second-strand synthesis on a single-stranded DNA template containing a primer-annealed double-stranded region, (b) filling-in reaction on a gapped duplex, and (c) polymerization reactions on nicked DNA which, depending on polymerases, may be a nick translation Pol I, path A) or a strand displacement synthesis (Pol Ik, path B). 

Various versions of uracil-based mutagenesis exist that differ in the preparation and use of dsDNA templates. In one variation, gapped duplex molecules are used as the target template for oligonucleotide-directed polymerization (14). The gapped duplex circular templates are generated by mixed annealing between... 

After synthesis, the RNA primers must be removed, gaps filled in, and the strands joined to give a linear, duplex DNA. New histones are added to the lagging strand (which is now a duplex) while the old histones remain with the leading strand. As the smoke slowly clears, we have a copy of the original DNA. 

Fills gaps in duplexes by stepwise addition of nucleotides to 3 ends Makes a DNA copy of an RNA molecule... 

RecA protein binds to single-stranded or gapped DNA. The complementary strand of the linear DNA pairs with a circular single strand. The other linear strand is displaced (left) or pairs with its complement in the circular duplex to yield a Holliday structure (right). 

The repair of stalled replication forks entails a coordinated transition from replication to recombination and back to replication. The recombination steps function to fill the DNA gap or rejoin the broken DNA branch to recreate the branched DNA structure at the replication fork. Lesions left behind in what is now duplex DNA are repaired by pathways such as base-excision or nucleotide-excision repair. Thus a wide range of enzymes encompassing every aspect of DNA metabolism ultimately take part in the repair of a stalled replication fork. This type of repair process is clearly a primary function of the homologous recombination system of every cell, and defects in recombinational DNA repair play an important role in human disease (Box 25-1). 

Figure 27-20 (A) Hypothetical replisome for concurrent replication of leading and lagging strands by a dimeric polymerase associated with helicase dnaB and a primosome. Open arrows indicate directions of movement of DNA, which is forming a loop as the polymerase fills a gap to complete an Okazaki fragment. The primase will then form a new primer and a new loop. From Komberg and Baker.265 (B) Electron micrograph of the primosome bound to covalently closed ( )X174 duplex replicative form. These enzymatically synthesized duplexes invariably contain a single primosome with one or two associated small DNA loops. From A. Komberg in Hubscher and Spadari,266 pp. 9,10.

The T-odd bacteriophages Tl, T3, T5, and T7 are medium-sized phage with linear duplex DNA genomes. Replication of linear DNA in these and in many other genomes presents a problem. Even if the RNA primer segment is made at the very 3 end of the template strand, there will be a gap in the final replicated strand when the primer is digested out. Since there is no known enzyme that will add to the 3 end of a chain, this gap will remain unfilled. The problem is solved by terminal redundancy, the presence of a common 260-nucleotide... 

The cross-stranded structure shown in Fig. 27-22 can be formed with all base pairs in both duplexes intact.526 527 All that is required is formation of a nick in each of the two polynucleotide chains and a rejoining of the backbones across the close gap between the duplexes. This model also accounts for the cutting of the two crossed strands at exactly equivalent points to terminate the process. Various mechanisms of recombination exist, and most make use of the key... 

Reactions catalyzed by purified recA protein in vitro. RecA catalyzes a number of different reactions between DNA strands, all of them involving the unwinding and winding of base-paired structures, (a) D-loop formation by interaction between supercoiled circular duplex DNA and single-stranded DNA. (b) Strand exchange between a gapped circular duplex structure and a linear duplex structure, (c) Complex formation between two helices, one of which is gapped. 

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