Exonucleolytic attack

The primary transcripts generated by RNA polymerase II—one of three distinct nuclear DNA-depen-dent RNA polymerases in eukaryotes—are promptly capped by 7-methylguanosine triphosphate caps (Figure 35-10) that persist and eventually appear on the 5 end of mature cytoplasmic mRNA. These caps are necessary for the subsequent processing of the primary transcript to mRNA, for the translation of the mRNA, and for protection of the mRNA against exonucleolytic attack. 

Trilling and Aposhian have partially purified a DNase from extracts of B. subtilis infected with phage SP-3 (35). This enzyme requires magnesium ion and show s optimal activity between pH 7.8 and 8.9 in Tris buffers. It is highly specific for denatured DNA and appears to catalyze a unique type of exonucleolytic attack beginning at the 5 end of the chain which sequentially releases dinucleotides. Neither mono-... 

To eliminate exonucleolytic attack as a cause of mRNA degradation we followed the fate of mRNA stably attached to ribosomes. If an inhibitor of polypeptide chain elongation is added to the incubations to prevent ribosome movement along mRNA, breakdown of polysomes can only result from endonucleolytic cleavage of the mRNA strand connecting ribosomes. Figure 2 shows... 

The enzyme begins its exonucleolytic attack at the 5 terminus of DNA, liberating almost entirely 5 -dNMP (11). The 5 -exonuclease hydrolyzes 5 -phosphoryl-and 5 -OH-terminated DNA at equal rates (12). The enzyme releases a dinucleoside monophosphate from the 5 -OH terminus and NTP from the 5 -triphosphate terminus (13). In fact, the gene 6 nuclease has been shown, like the S nuclease of E. coli DNA Pol I, to function on model substrates as a structure-specific endonuclease (46). The gene 6 nuclease also degrades RNA in a variety of RNA DNA hybrids and thus has an RNase H activity. 

An ATP-dependent DNase has been partially purified from extracts of E. coli by Oishi (86) and Barbour and Clark (37). It shows an absolute requirement for magnesium or manganese ion and has a broad pH optimum ranging from pH 7.5 to 9.5. The partially purified enzyme preferentially degrades native DNA (including glucosylated T4 DNA) and has an almost absolute requirement for ATP or dATP. Current preparations of the enzyme are also active on denatured DNA however, there is only a slight stimulation of hydrolysis by added ATP. This latter activity may therefore represent some contamination with exonuclease I. The mode of attack is stated to be exonucleolytic. 

Bal31 attacks predominantly from the 3 termini. The nucleolytic action on protruding 5 single strands is presumed to involve two successive activities initially an endonuclease activity and subsequently the exonucleolytic activity. During the course of Bal31 digestions of either ssDNA or dsDNA, however, possible intermediary oligonucleotide products have not been observed. 

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