JUNCTION RIBONUCLEASE - AN ACTIVITY IN OKAZAKI FRAGMENT PROCESSING

The initiator RNAs of mammalian Okazaki fragments are thought to be re moved by RNase HI and the 5'-3' flap endonuclease (FEN1), Earlier evid ence indicated that the cleavage site of RNase HI is 5' of the last ri bonucleotide at the RNA-DNA junction on an Okazaki substrate, In curre nt work, highly purified calf RNase HI makes this exact cleavage in Ok azaki fragments containing mismatches that distort the hybrid structur e of the heteroduplex. Furthermore, even fully unannealed Okazaki frag ments were cleaved, Clearly, the enzyme recognizes the transition from RNA to DNA on a single-stranded substrate and not the RNA/DNA heterod uplex structure, We have named this junction RNase activity, This acti vity exactly comigrates with RNase HI activity during purification str ongly suggesting that both activities reside in the same enzyme, After junction cleavage, FEN1 removes the remaining ribonucleotide, Because FEN1 prefers a substrate with a single-stranded 5'-flap structure, th e single-stranded activity of junction RNase suggests that Okazaki fra gments are displaced to form a 5'-tail prior to cleavage by both nucle ases.