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. 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. INTRODUCTIONA 5′ to 3′ exonuclease purified from calf has been shown to cooperate functionally with calf DNA polymerase εto perform nick translation (Siegal et al., 1992). In this reaction, exonucleolytic activity on a downstream primer required synthesis from an upstream primer. The exonuclease was later found to be much more active on a nicked compared with a gapped double-stranded DNA substrate (Murante et al., 1994). This suggests that stimulation of nuclease activity by DNA synthesis resulted from continuous generation of the favored substrate.The calf 5′ to 3′ exonuclease has been implicated in processing of Okazaki fragments (Turchi et al., 1994). A DNA template, primed with an oligoribonucleotide that had been extended at the 3′ end with DNA, was used as a model Okazaki fragment. Calf RNase H1 removed the initiator RNA with a single cut at the 5′ side of the ribonucleotide at the RNA-DNA junction. The 5′ to 3′ exonuclease then could remove the remaining ribonucleotide, creating a 5′ DNA. Extension of an upstream primer by polymerization in the presence of DNA ligase I allowed the two primers to be joined, as expected on the lagging replication fork strand in vivo.In addition to the above activities, the 5′ to 3′ exonuclease could also cleave endonucleolytically (Murante et al., 1994). The substrate for this reaction was a DNA primer-template, having a downstream primer with a noncomplementary, unannealed region at the 5′ end. Additionally, an upstream primer was annealed directly adjacent to the first annealed nucleotide of the downstream primer. Endonucleolytic cleavage removed the unannealed region as an intact segment. Cleavage occurred either at the point of annealing to make a nicked substrate or one nucleotide downstream to make a 1-nucleotide gap. The 5′ to 3′ exonuclease domains of Escherichia coli DNA polymerase I and Thermus aquaticus (Taq) DNA polymerase (Lyamichev et al., 1993) are functionally homologous to the calf nuclease. The homologous nuclease has also been purified from murine cells and designated cca nuclease (Goulian et al., 1990) and flap endonuclease (FEN-1) (Harrington and Lieber, 1994). The corresponding HeLa cell nuclease has been purified (Ishimi et al., 1988; Waga et al., 1994; Robins et al., 1994; Murray et al., 1994), and called maturation factor I (Waga et al., 1994) and DNase IV (Robins et al., 1994). The homolog of this mammalian protein has been identified in Saccharomyces cerevisiae and named the RTH-1 nuclease (Sommers et al., 1995; Johnson et al., 1995). Knockout mutants are temperature-sensitive for DNA replication and repair of methylmethane sulfonate lesions (Sommers et al., 1995). RTH-1 nuclease was also found to be necessary for the stability of DNA repeats in yeast, suggesting that mutation of the human counterpart could impair the mismatch repair pathway that protects from colorectal cancer (Johnson et al., 1995).In this report, we explore the substrate structure requirements for endonuclease activity of the calf nuclease. We show that the nuclease must slide over the 5′ end of the unannealed tail of the primer and then along the single-stranded tail to the point of cleavage. The sliding action is bidirectional. The presence of a primer or protein on the tail prevents entry of the nuclease or traps a nuclease that is already on the tail. Cleavage is observed on tails with covalent adducts on nucleotides either at the 5′ end or within the tail. A model is proposed that has implications for the biological role of this unusual nuclease.DISCUSSIONThe calf 5′ to 3′ exo/endonuclease is a member of the RTH-1 class of eukaryotic nucleases that perform structure-specific cleavage of DNA and have important roles in DNA replication and repair. The substrate for endonuclease activity is a primer-template in which the primer has a noncomplementary, unannealed 5′ end region. Here we show that in order to catalyze cleavage, the nuclease must slide over the 5′ end of the unannealed tail, move down the length of the tail to the point of annealing with the template, and then carry out cleavage. The sliding process is bidirectional, such that nucleases can also slide off of the tail at the 5′ end. Evidence is also presented that the nuclease binds with highest stability to the point of cleavage. The presence of biotin adducts on the tail do not affect either recognition of the 5′ end or the sliding process. However, protein bound to the tail, either SSB or streptavidin, has an inhibitory effect on cleavage, presumably by blocking the movement of the nuclease to the site of cleavage. The steps taken by the nuclease to carry out this process are depicted in Fig. 8.Biochemical and genetic evidence suggests that nucleases of this class are components of the DNA replication machinery. Studies of simian virus 40 DNA replication in vitro indicated the requirement of the human 5′ to 3′ exo/endonuclease in the removal of the RNA primer before joining of viral lagging strand DNA segments (Ishimi et al., 1988; Waga et al., 1994). The mouse nuclease was able to partially remove RNA from initiator RNA primers made and elongated with DNA by DNA polymerase α in vitro (Goulian et al., 1990). Using a model Okazaki fragment substrate consisting of an initiator RNA elongated by DNA and annealed to a template, we showed that two nucleases can effect complete RNA removal (Turchi et al., 1994). Mammalian RNase HI cleaves off the initiator RNA as an intact segment, leaving only a single ribonucleotide at the RNA-DNA junction (Turchi et al., 1994; Huang et al., 1994). The 5′ to 3′ exo/endonuclease can then remove the last ribonucleotide. Null mutants of the counterpart nuclease in S. cerevisiae, the RTH-1 nuclease, are temperature-sensitive for DNA replication (Sommers et al., 1995). The appearance of dumbbell-shaped cells with a nucleus at the isthmus as the terminal phenotype is characteristic of mutations in DNA polymerases δ and ε(reviewed in Bambara and Huang(1995)). The RTH-1 mutation causes hyper-recombination, symptomatic of the presence of long-lived breaks in the chromosome (Sommers et al., 1995). These would be expected if joining of Okazaki fragments was delayed by the mutation.The sensitivity of the RTH-1 mutants to damage by the agent methylmethane sulfonate (Sommers et al., 1995), which adducts to bases, suggests that the RTH-1 class of nucleases participate in repair of base damage. The demonstration that null mutants in RTH-1 increase the instability of simple repetitive DNA indicates a role for this nuclease in the mismatch repair pathway (Johnson et al., 1995). Our results show that biotinylation of nucleotides on a tail do not inhibit the movement of the nuclease to its site of cleavage. This suggests that in vivo the nuclease can traverse an unannealed, damaged segment of DNA and cut it away from the chromosome. Because the nuclease does not have to cut at the damaged nucleotide to accomplish this process, it should be able to remove even modified nucleotides that would not be substrates for cleavage attempted at the modified site.Our results show that the nuclease cannot enter the tail strand except at the 5′ end. Because the RTH-1 class nucleases are expected to be present at the replication fork, this property would be expected to protect the lagging strand template from unintentional cleavage. There is a risk of such cleavage because the extension of the leading strand primer continuously generates a structure with most of the characteristics of the substrate for RTH-1 endonuclease activity, at the point where the parental strands separate. In this structure, the lagging strand template would be the equivalent of the single-stranded tail in our model substrates, except that the lagging strand template does not have a nearby 5′ end. The absence of this single feature prevents endonucleolytic cleavage.Properties of the calf 5′ to 3′ exo/endonuclease suggest another means by which it can participate in the removal of the initiator RNA segments of Okazaki fragments. It could remove this RNA by endonucleolytic cleavage of the Okazaki fragment in the region of DNA just downstream of the initiator RNA. DNA polymerase εhas been proposed to be responsible for completion of the extension of Okazaki fragments in vivo (reviewed in Bambara and Huang(1995)). This polymerase partially copurifies with DNA helicase E (Siegal et al., 1992). The Ku autoantigen is also a helicase that copurifies with human DNA polymerase ε(Hwang et al., 1995), suggesting that helicase E is the calf Ku protein. Helicase E moves in a 3′ to 5′ direction on the template and so can displace primers ahead of the advancing polymerase. It is possible that the polymerase, helicase, and RTH-1 class nuclease collaborate in a pathway for removal of initiator RNAs of Okazaki fragments.In summary, the calf 5′ to 3′ exo/endonuclease can cleave off the 5′ unannealed tail of a primer annealed to a template by cutting near the point of complementarity and releasing the tail as an intact segment. Here we show that to catalyze this reaction, the nuclease must enter the tail at the 5′ end and slide to the point of cleavage. This unique mechanism of action is consistent with a proposed role for the nuclease in removal of the initiator primers from Okazaki fragments during DNA replication and in cleaving off segments of damaged DNA during DNA repair. The yeast counterpart nuclease RTH-1 has been shown to be necessary to maintain stability of repetitive DNA and apparently is involved in the MSH2-MLH1-PMS1 mismatch repair pathway (Johnson et al., 1995). Mutations in the mammalian mismatch repair pathway are associated with colorectal cancers (Reenan and Kolodner, 1992; Prolla et al., 1994). This suggests that the human RTH-1 counterpart applies its unique cleavage specificity for repair surveillance that prevents colorectal cancer. INTRODUCTIONA 5′ to 3′ exonuclease purified from calf has been shown to cooperate functionally with calf DNA polymerase εto perform nick translation (Siegal et al., 1992). In this reaction, exonucleolytic activity on a downstream primer required synthesis from an upstream primer. The exonuclease was later found to be much more active on a nicked compared with a gapped double-stranded DNA substrate (Murante et al., 1994). This suggests that stimulation of nuclease activity by DNA synthesis resulted from continuous generation of the favored substrate.The calf 5′ to 3′ exonuclease has been implicated in processing of Okazaki fragments (Turchi et al., 1994). A DNA template, primed with an oligoribonucleotide that had been extended at the 3′ end with DNA, was used as a model Okazaki fragment. Calf RNase H1 removed the initiator RNA with a single cut at the 5′ side of the ribonucleotide at the RNA-DNA junction. The 5′ to 3′ exonuclease then could remove the remaining ribonucleotide, creating a 5′ DNA. Extension of an upstream primer by polymerization in the presence of DNA ligase I allowed the two primers to be joined, as expected on the lagging replication fork strand in vivo.In addition to the above activities, the 5′ to 3′ exonuclease could also cleave endonucleolytically (Murante et al., 1994). The substrate for this reaction was a DNA primer-template, having a downstream primer with a noncomplementary, unannealed region at the 5′ end. Additionally, an upstream primer was annealed directly adjacent to the first annealed nucleotide of the downstream primer. Endonucleolytic cleavage removed the unannealed region as an intact segment. Cleavage occurred either at the point of annealing to make a nicked substrate or one nucleotide downstream to make a 1-nucleotide gap. The 5′ to 3′ exonuclease domains of Escherichia coli DNA polymerase I and Thermus aquaticus (Taq) DNA polymerase (Lyamichev et al., 1993) are functionally homologous to the calf nuclease. The homologous nuclease has also been purified from murine cells and designated cca nuclease (Goulian et al., 1990) and flap endonuclease (FEN-1) (Harrington and Lieber, 1994). The corresponding HeLa cell nuclease has been purified (Ishimi et al., 1988; Waga et al., 1994; Robins et al., 1994; Murray et al., 1994), and called maturation factor I (Waga et al., 1994) and DNase IV (Robins et al., 1994). The homolog of this mammalian protein has been identified in Saccharomyces cerevisiae and named the RTH-1 nuclease (Sommers et al., 1995; Johnson et al., 1995). Knockout mutants are temperature-sensitive for DNA replication and repair of methylmethane sulfonate lesions (Sommers et al., 1995). RTH-1 nuclease was also found to be necessary for the stability of DNA repeats in yeast, suggesting that mutation of the human counterpart could impair the mismatch repair pathway that protects from colorectal cancer (Johnson et al., 1995).In this report, we explore the substrate structure requirements for endonuclease activity of the calf nuclease. We show that the nuclease must slide over the 5′ end of the unannealed tail of the primer and then along the single-stranded tail to the point of cleavage. The sliding action is bidirectional. The presence of a primer or protein on the tail prevents entry of the nuclease or traps a nuclease that is already on the tail. Cleavage is observed on tails with covalent adducts on nucleotides either at the 5′ end or within the tail. A model is proposed that has implications for the biological role of this unusual nuclease.