The multiple activities of Escherichia coli endonuclease IV and the extreme lability of 5′-terminal base-free deoxyribose 5-phosphates

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Abstract
Escherichia coli endonuclease IV hydrolyses the C(3'')-O-P bond 5'' to a 3''-terminal base-free deoxyribose. It also hydrolyses the C(3'')-O-P bond 5'' to a 3''-terminal base-free 2'',3''-unsaturated sugar produced by nicking 3'' to an AP (apurinic or apyrimidinic) site by .beta.-elimination; this explains why the unproductive end produced by .beta.-elimination is converted by the enzyme into a 3''-OH end able to prime DNA synthesis. The action of E. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3'')-O-P bond 5'' to the AP site is hydrolysed, but in a second step the 5''-terminal base-free deoxyribose 5''-phosphate is lost. This loss is due to a spontaneous .beta.-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3'')-O-P bond 3'' to a 5''-terminal AP site contrasts with the relative stability of the same bond 3'' to an internal AP site; in the absence of .beta.-elimination catalysts, at 37.degree.C the half-life of the former is about 2h and that of the latter 200 h. The extreme lability of a 5''-terminal AP site means that, after nicking 5'' to an AP site with an AP endonuclease, in principle no 5'' .fwdarw. 3'' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'' .fwdarw. 3'' exonuclease activity (Klenow polymerase or rat liver DNA polymerase .beta.) and a DNA ligase. Catalysts of .beta.-elimination, such as spermine, can drastically shorten the already brief half-life of a 5''-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3''-phosphoglycollatase and also a 3''-phosphatase. The 3''-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a .beta..delta.-elimination reaction.