Junction ribonuclease: an activity in Okazaki fragment processing.

The initiator RNAs of mammalian Okazaki fragments are thought to be removed by RNase HI and the 5'-3' flap endonuclease (FEN1). Earlier evidence indicated that the cleavage site of RNase HI is 5' of the last ribonucleotide at the RNA-DNA junction on an Okazaki substrate. In current work, highly purified calf RNase HI makes this exact cleavage in Okazaki fragments containing mismatches that distort the hybrid structure of the heteroduplex. Furthermore, even fully unannealed Okazaki fragments were cleaved. Clearly, the enzyme recognizes the transition from RNA to DNA on a single-stranded substrate and not the RNA/DNA heteroduplex structure. We have named this junction RNase activity. This activity exactly comigrates with RNase HI activity during purification strongly 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, the single-stranded activity of junction RNase suggests that Okazaki fragments are displaced to form a 5'-tail prior to cleavage by both nucleases.

Creation and removal of embedded ribonucleotides in chromosomal DNA during mammalian Okazaki fragment processing.

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.

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 initiator RNA from Okazaki fragments. Cleavage requires that the RNA be displaced to form an unannealed single-stranded 5'-tail or flap structure. On substrates with RNA-initiated primers, DNA oligomers that competed with the RNA for template binding simulated strand displacement synthesis from an upstream Okazaki fragment. This allowed cutting of displaced RNA segments by RTH-1 nuclease. Requirements for the reaction also were examined on substrates in which the tail was unannealed because it was intentionally mispaired. On both types of substrate, the nuclease slides over the RNA region from the 5'-end and cleaves at the beginning of the annealed region, irrespective of whether ribo- or deoxyribonucleotides 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 nuclease by an upstream primer was not always observed, suggesting that not every site in the downstream Okazaki fragment is equally susceptible to cleavage during displacement synthesis in vivo. The biological role of the endonuclease activity of RTH-1 nuclease in Okazaki fragment processing is discussed.

Role of calf RTH-1 nuclease in removal of 5'-ribonucleotides during Okazaki fragment processing.

The role of the exonucleolytic activity of the calf 5' to 3' exo/endonuclease, a RAD2 homolog 1 (RTH-1) class nuclease, in lagging-strand DNA replication has been examined using model Okazaki fragment substrates. These substrates exemplify the situation in Okazaki fragment processing which occurs after the initiator RNA primer is cleaved off, and released intact, by calf RNase HI, leaving a single ribonucleotide at the 5' end of the RNA-DNA junction. This final RNA is then removed by the calf RTH-1 nuclease [Turchi et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9803-9807]. The cleavage specificity of calf RTH-1 nuclease for different junction ribonucleotides was compared. These were removed without the usual requirement of calf RTH-1 for an immediately adjacent upstream primer. In most cases, the presence of an upstream DNA or RNA primer, separated from the monoribonucleotide-DNA segment by either a nick or a gap, reduced the efficiency of removal of the monoribonucleotide compared to the removal seen with no upstream primer. Substrates in which the monoribonucleotide-DNA segment had been replaced by an oligomer of the same sequence but consisting entirely of DNA also exhibited upstream primer inhibition. Results with various sequences indicated that the upstream primer is generally inhibitory for ribonucleotide removal but is sometimes neutral. For deoxynucleotide removal it could be stimulatory, neutral, or inhibitory. Possible reasons for the unexpected lack of upstream primer dependence have been explored. The ratio of RNase HI to RTH-1 was also shown to be critical for both enzymes to work together efficiently. These results suggest that regions of upstream primer inhibition within the genome may play a role in determining the mechanism by which mammalian Okazaki fragments are processed.

Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage.

Calf 5' to 3' exo/endonuclease, the counterpart of the human FEN-1 and yeast RTH-1 nucleases, performs structure-specific cleavage of both RNA and DNA and is implicated in Okazaki fragment processing and DNA repair. The substrate for endonuclease activity is a primer annealed to a template but with a 5' unannealed tail. The results presented here demonstrate that the nuclease must enter the 5' end of the unannealed tail and then slide to the region of hybridization where the cleavage occurs. The presence of bound protein or a primer at any point on the single-stranded tail prevents cleavage. However, biotinylation of a nucleotide at the 5' end or internal to the tail does not prevent cleavage. The sliding process is bidirectional. If the nuclease slides onto the tail, later binding of a primer to the tail traps the nuclease between the primer binding site and the cleavage site, preventing the nuclease from departing from the 5' end. A model for 5' entry, sliding, and cleavage is presented. The possible role of this unusual mechanism in Okazaki fragment processing, DNA repair, and protection of the replication fork from inappropriate endonucleolytic cleavage is presented.

Enzymatic completion of mammalian lagging-strand DNA replication.

Using purified proteins from calf and a synthetic substrate, we have reconstituted the enzymatic reactions required for mammalian Okazaki fragment processing in vitro. The required reactions are removal of initiator RNA, synthesis from an upstream fragment to generate a nick, and then ligation. With our substrate, RNase H type I (RNase HI) makes a single cut in the initiator RNA, one nucleotide 5' of the RNA-DNA junction. The double strand specific 5' to 3' exonuclease removes the remaining monoribonucleotide. After dissociation of cleaved RNA, synthesis by DNA polymerase generates a nick, which is then sealed by DNA ligase I. The unique specificities of the two nucleases for primers with initiator RNA strongly suggest that they perform the same reactions in vivo.

The calf 5'- to 3'-exonuclease is also an endonuclease with both activities dependent on primers annealed upstream of the point of cleavage.

The catalytic activity of the calf thymus 5'- to 3'-exonuclease was measured on substrates consisting of two primers annealed adjacent to each other on a template. Exonucleolytic degradation of the downstream primer is very slow if the primers are separated by a gap of one nucleotide or if no upstream primer is present. When only a nick separates the primers, degradation is rapid. This suggests that the nuclease is designed to work with calf DNA polymerases such that synthesis from an upstream primer creates the favored nuclease substrate. Nuclease action then destroys the substrate, but it is regenerated by further polymerization. This process, termed nick translation, is necessary for both DNA replication and repair. If the downstream primer has an unannealed 5'-region, that region is removed by an endonuclease activity residing in the same enzyme. Efficient endonuclease action also requires an upstream primer that is annealed such that its 3'-end is directly adjacent to the annealed region of the downstream primer. This reaction is likely to be important for removal of DNA segments that are damaged such that exonuclease cleavage of the damaged site is not possible.

DNA substrate specificity of DNA helicase E from calf thymus.

DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair.