Creation and Removal of Embedded Ribonucleotides in Chromosomal DNA during Mammalian Okazaki Fragment Processing

Abstract

Mammalian RNase HI has been shown to specifically cleave the initiator RNA of Okazaki fragments at the RNA-DNA junction, leaving a single ribonucleotide attached to the 5'-end of the downstream DNA segment. This monoribonucleotide can then be removed by the mammalian 5'- to 3'-exo-/endonuclease, a RAD2 homolog-1 (RTH-1) class nuclease, also known as flap endonuclease-1 (FEN-1). Although FEN-1/RTH-1 nuclease often requires an upstream primer for efficient activity, the presence of an upstream primer is usually inhibitory or neutral for removal of this 5'-monoribonucleotide. Using model Okazaki fragment substrates, we found that DNA ligase I can seal a 5'-monoribonucleotide into DNA. When both ligase and FEN-1/RTH-1 were present simultaneously, some of the 5'-monoribonucleotides were ligated into DNA, while others were released. Thus, a 5'-monoribonucleotide, particularly one that is made resistant to FEN-1/RTH-1-directed cleavage by extension of an inhibitory upstream primer, can be ligated into the chromosome, despite the presence of FEN-1/RTH-1 nuclease. DNA ligase I was able to seal different monoribonucleotides into the DNA for all substrates tested, with an efficiency of 1-13% that of ligating DNA. These embedded monoribonucleotides can be removed by the combined action of RNase HI, cutting on the 5'-side, and FEN-1/RTH-1 nuclease, cleaving on the 3'-side. After FEN-1/RTH-1 action and extension by polymerization, DNA ligase I can join the entirely DNA strands to complete repair.