Replacement synthesis exonuclease-polymerase activity

Labeling through sequential exonuclease-polymerase activities ( replacement synthesis )... 

FIGURE 25-9 Nick translation. In this process, an RNA or DNA strand paired to a DNA template is simultaneously degraded by the 5 —>3 exonuclease activity of DNA polymerase I and replaced by the polymerase activity of the same enzyme. These activities have a role in both DNA repair and the removal of RNA primers during replication (both described later). The strand of nucleic acid to be removed (either DNA or RNA) is shown in green, the replacement strand in red. DNA synthesis begins at a nick (a broken phosphodiester bond, leaving a free 3 hydroxyl and a free 5 phosphate). Polymerase I extends the nontemplate DNA strand and moves the nick along the DNA—a process called nick translation. A nick remains where DNA polymerase I dissociates, and is later sealed by another enzyme. 

DNA polymerase I then uses its polymerizing and 5 to 3 exonuclease activities to remove the RNA primer and fill in this sequence with DNA. Because Pol I is not very processive, it falls off the lagging strand after a relatively short-length synthesis. DNA polymerases can t seal up the nicks that result from the replacement of RNA primers with DNA. Instead, another enzyme, DNA ligase, seals off the nicks by using high energy phosphodiester bonds in ATP or NAD to join a free 3 hydroxyl with an adjacent 5 phosphate. 

The activity will be similar to the replacement of an RNA primer with DNA by DNA polymerase I. One makes use of the combined 5 —> 3 exonuclease and 5 —> 3 polymerase activities of DNA polymerase I. From the point of the internal nick (of only one strand) by the endonuclease, polymerase I will extend the free 3 -OH using radioactive dNTPs while at the same time digesting from the internal 5 -phosphate to make room for the newly synthesized DNA. The result is a nick translation event in which an unlabeled portion of one DNA strand is replaced with a radioactive stretch of DNA. (Over the section of new synthesis, only one strand becomes labeled. The strand used as the template remains unlabeled.)... 

The 3 end of DNA can be labeled more extensively by means of replacement synthesis (32). In this method, duplex DNA is first digested in the absence of dNTPs by the 3 — 5 -exonuclease activity, e.g., up to 30-40% of the length from each end. (Note that about 50% digestion will lead to dissociation of the two single-stranded halves and result in a loss of the template due to rapid degradation.) Subsequently, four dNTPs containing one [a- P]dNTP are added to the reaction mixture in order for the polymerase to extend the 3 ends to the length of the template. 

FIGURE 25-15 Final steps in the synthesis of lagging strand segments. RNA primers in the lagging strand are removed by the 5 —>3 exonuclease activity of DNA polymerase I and replaced with DNA by the same enzyme. The remaining nick is sealed by DNA ligase. The role of ATP or NAD+ is shown in Figure 25-16. 

While the replacement DNA synthesis method for 3 -end labeling requires both the 3 - 5 exonuclease and DNA polymerase functions of DNA polymerases, the activity of the 5 - 3 DNA polymerase alone is sufficient to produce radiolabeled blunt-ended DNA frj ents with substrates containing protruding 5 termini. For example, the recessed 3 terminus shown in Fig. 1C can be extended by DNA polymerase in the presence of dXTP and dCTP to yield the blunt-ended DNA fragment indicated. 

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