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.During cellular DNA replication, the leading strand is synthesized continuously in the direction of replication fork propagation. An antiparallel template is used for synthesis of the lagging strand, which therefore must be made as a series of discontinuous segments called Okazaki fragments. As the replication fork opens, new upstream fragments are initiated. Each fragment must be independently primed with initiator RNA, which is later removed, prior to joining of the segments into one continuous strand (1). In eukaryotes, initiator RNA removal is achieved, as reviewed in Bambara et al. (2), by the combined action of two nucleases, RNase HI and a 5Ј-to 3Ј-exo-/endonuclease, called RTH-1 or FEN-1 (3-7). RNase HI makes a structure-specific cleavage, releasing the initiator RNA as an intact segment but leaving a single ribonucleotide on the 5Ј-end of the downstream DNA. FEN-1/RTH-1 nuclease can then remove this monoribonucleotide (8). Although FEN-1/RTH-1 cleavage often requires an upstream primer for stimulation, the presence of an upstream primer is sometimes neutral or even inhibitory, especially for removal of these monoribonucleotides (9). If an upstream primer approaches before FEN-1/RTH-1 action and inhibition at the particular junction is significant, Okazaki fragment processing may be halted, leaving a nick in the chromosome, just upstream of a single ribonucleotide. In such a situation, processing might still be completed via the endonucleolytic activity of FEN-1/RTH-1 nuclease (9). However, if DNA ligase is able to seal such monoribonuc...