CALF RTH-1 NUCLEASE CAN REMOVE THE INITIATOR RNAS OF OKAZAKI FRAGMENTS BY ENDONUCLEASE ACTIVITY

In eukaryotes, the endonucleolytic activity of the calf RTH-1 class 5' - to 3'-exo/endonuclease can function without RNase H1 to remove initi ator RNA from Okazaki fragments, Cleavage requires that the RNA be dis placed to form an unannealed single-stranded 5'-tail or flap structure , On substrates with RNA-initiated primers, DNA oligomers that compete d with the RNA for template binding simulated strand displacement syn thesis from an upstream Okazaki fragment, This allowed cutting of disp laced RNA segments by RTH-1 nuclease, Requirements for the reaction al so were examined on substrates in which the tail was unannealed becaus e it was intentionally mispaired. On both types of substrate, the nucl ease slides over the RNA region from the 5'-end and cleaves at the beg inning of the annealed region, irrespective of whether ribo- or deoxyr ibonucleotides are at the cleavage site. Presence of a triphosphate or a 7-methyl 3'G5' ppp5' G cap structure at the 5'-end of the RNA does not affect cleavage, The previously reported stimulation of the nuclea se by an upstream primer was not always observed, suggesting that not every site in the downstream Okazaki fragment is equally susceptible t o cleavage during displacement synthesis in vice, The biological role of the endonuclease activity of RTH-1 nuclease in Okazaki fragment pro cessing is discussed.