Cleavage of substrates with mismatched nucleotides by flap endonuclease-1 - Implications for mammalian Okazaki fragment processing

Flap endonuclease-l (FEN1) is proposed to participate in removal of the ini tiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA FEN1 removes this ribonucleotide exonucleolytically. In the othe r pathway, FEN1 removes the entire primer endonucleolytically after displac ement of the 5'-end region of the Okazaki fragment. Cleavage would occur be yond the RNA, a short distance into the DNA, The initiator RNA and an adjac ent short region of DNA are synthesized by DNA polymerase alpha/primase. Be cause the fidelity of DNA polymerase a is lower than that of the DNA polyme rases that complete DNA extension, mismatches occur relatively frequently n ear the 5'-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleoti des from the 5'-end of an annealed DNA strand change the pattern of FEN1 cl eavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propo se that a mismatch destabilizes the helical structure over a nearby area. T his allows FEN1 to cleave more efficiently, facilitating removal of the mis match. If mismatches were not introduced during synthesis of the Okazaki fr agment, helical disruption would not occur, nor would unnecessary degradati on of the 5'-end of the fragment.