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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 8, Issue of February 21, pp. 46474650, 1997 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
subunit () form a complex at the primer terminus. is at the 3-end and immediately behind are the other subunits contacting the double-stranded region of the primer-template (28). By both analogy and preliminary experimental evidence, RFC is retained in the complex of PCNA and DNA pol .2 Synthesis of the Lagging Strand Priming by DNA pol and switching to DNA pol occur in essentially the same way on both the lagging and leading strand of SV40 (29, 30) as illustrated in Fig. 1A. In fact, polymerase switching during synthesis of Okazaki fragments was found to be a necessary prerequisite for complete gap filling (30). Analysis of SV40 replication intermediates indicates that priming on the lagging strand is very frequent, with initial placement of primers 50 nucleotides apart (31). Okazaki fragment intermediates consist of initiator RNAs averaging about 10 nucleotides in length, extended with 10 20 additional deoxyribonucleotides (31). Because Okazaki fragment intermediates are made in the absence of ATP, and RFC requires ATP for polymerase switching, the deoxyribonucleotides of the intermediates appear to be added by DNA pol prior to the switch. After loading of PCNA and DNA pol , an additional 10 20 nucleotides are added prior to the position of the next downstream initiator RNA primer. There must be further extension of the upstream primer during or after removal of the initiator RNA and possibly nick translation synthesis through the first deoxyribonucleotides of the downstream primer. DNA pol should then dissociate. Results from the homologous E. coli system for DNA pol III (32) indicate that encountering a downstream primer induces the dissociation of the processive complex. If eukaryotes operate similarly, the necessary frequent dissociations of DNA pol would be promoted by contact with the downstream primer. Numerous RNA primers must be removed. The action of two nucleases is sufficient to perform this task (33). RNase H1 endonucleolytically cleaves the initiator RNA one nucleotide upstream of the RNA-DNA junction. The initiator RNA is removed intact leaving a single ribonucleotide on the downstream DNA portion of the Okazaki fragment. RNase H1 cleavage leaves the junctional ribonucleotide irrespective of the length of the initiator RNA. The position of cleavage is not affected by either the RNA or DNA sequence around the cleavage site. In fact, cleavage remains specific even if mismatched nucleotides are present at positions at and near the RNA-DNA junction.3 This indicates that RNase H1 recognizes the junction and not the intermediate structure between the A-form helix of the RNA-DNA duplex and the B-form helix of the DNA duplex. The structure of these regions is likely distorted in the mismatched substrates. The remaining ribonucleotide is removed (30, 33) by FEN1/RTH1 (30, 34 37), which contains both an exonucleolytic and endonucleolytic capability (38, 39). As an exonuclease, it is specific for DNA or 5-RNA terminated DNA primers annealed to templates (40). Depending on the sequence of the region, it may be stimulated, unaffected, or inhibited by the presence of a primer bound immediately upstream of the cleavage site (40). Removal of the junctional ribonucleotide is generally inhibited by an upstream primer. This suggests that the initiator RNA removed by RNase H1 dissociates before FEN1/RTH1-directed cleavage of the junctional ribonucleotide. During synthesis, the extension of the upstream primer by polymerization could stimulate nick translation. When a sequence is encountered such that the upstream primer is inhibitory, nick translation would stop, allowing ligation of the Okazaki fragments. We found that the simultaneous action of a DNA polymerase, RNase H1, FEN1/RTH1, and DNA ligase I results in correct Okazaki fragment processing in vitro (41) as depicted in Fig. 1B.
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FIG. 2. Removal of a displaced Okazaki initiator RNA by FEN1/ RTH1 nuclease. As synthesis of the upstream Okazaki fragment is completed, DNA polymerase with a DNA helicase displaces both RNA and DNA of the downstream fragment, generating an unannealed 5-tail. FEN1/RTH1 binds and tracks over the 5-tail. Then, FEN1/RTH1 endonucleolytically cleaves the 5-tail removing the entire RNA primer.
RTH1 could compensate for defective or absent RNase H1. In the absence of FEN1/RTH1, both pathways would fail, reverting to a third alternative pathway inefficient enough to produce the temperature-sensitive phenotype. Recent discovery of at least one additional gene in yeast that encodes a nuclease with a similar structure to FEN1/RTH1 (43, 44) suggests the possibility of a third pathway for RNA primer removal. Possible Role of the Endonuclease Activity of the FEN1/RTH1 Nuclease in Initiator RNA Removal The mammalian FEN1/RTH1 class nucleases also contain a unique, structure-specific endonucleolytic activity (38, 39). A favored substrate consists of two primers on a template having the annealed portions directly adjacent to each other. However, the 5-end of the downstream primer forms an unannealed 5-tail. Then, FEN1/RTH1 could cleave the tail endonucleolytically in the DNA region downstream of the initiator RNA. After the gap between the remaining DNA portions of the two primers is filled, ligation would complete Okazaki fragment processing. Endonucleolytic cleavage by FEN1/RTH1 occurs by a unique mechanism. The nuclease slides over the 5-end of the unannealed tail and traverses the entire length of the tail before arriving at the point of cleavage near the annealing point of the tail (39, 45). One of the most definitive experiments demonstrating an obligatory sliding mechanism involved use of a tail 73 nucleotides long. Annealing of a 20-nucleotide-long primer anywhere on the tail inhibited cleavage (46). Additionally, modification of the most 5-nucleotide with a biotin-containing side chain, followed by binding of streptavidin, prevented entry of the nuclease and cleavage (46). Calf FEN1/RTH1 readily recognizes the triphosphorylated 5end region of a displaced initiator RNA and cleaves in the downstream DNA (47). Surprisingly, several of the substrates used in this study were cleaved in the absence of a nick-like structure. Influences of nucleotide sequence on nick-dependent stimulation and specificity of cleavage were examined; however, no specific criteria could be discerned (47). Overall, results demonstrate that the endonucleolytic activity of the nuclease is capable of bypassing the need for RNase H1 in Okazaki fragment processing. The proposed cleavage mechanism of a displaced Okazaki fragment by FEN1/RTH1 nuclease is shown in Fig. 2. The structure of the mammalian 53-exo/endonuclease has not yet been determined. However, eukaryotic and prokaryotic nucleases of this type have homologous sequences (48). Recently, the x-ray crystal structures of three of the prokaryotic nucleases, Taq 5-exonuclease (49), the bacteriophage T5 53-exo/endonuclease (50), and the bacteriophage T4 5-nuclease (also called RNase H) (51), were determined. While the overall structures are similar, Ceska et al. (50) specifically pointed out an arch shape in the T5 nuclease, rising above the globular main section of the protein. Two -helical structures make up the supports of the arch, and the keystone region is an area of random coil. Working from known mechanistic information about this class of nucleases, Ceska et al. (50) proposed a way for the nuclease to interact with its substrate. Their
FIG. 1. DNA polymerase switching and processing of an Okazaki fragment on the lagging strand. A, as the DNA helicase promotes unwinding at the replication fork, DNA pol with RFC and PCNA synthesizes DNA on the leading strand. DNA pol initiates synthesis on the lagging strand by generating an RNA primer (red segment) followed by a short segment of DNA. Then, RFC and PCNA load a second DNA polymerase ( or ) to continue synthesis of the Okazaki fragment. B, as DNA pol approaches the downstream Okazaki fragment, cleavage by RNase H1 removes the initiator RNA primer leaving a single 5-ribonucleotide. Then, FEN1/RTH1 removes the 5-ribonucleotide. The resulting nick is sealed by DNA ligase.
Alternative Pathways for Okazaki Fragment Processing Genetic analyses in yeast suggest that there are alternative means of initiator RNA removal. Null mutants of the primary RNase H in yeast are not significantly defective in DNA replication.4 One possibility is that the yeast RNase H is not equivalent to mammalian RNase H1. Alternatively, there is an efficient second pathway for RNA removal that does not require RNase H. Null mutants of FEN1/RTH1 in yeast are temperature-sensitive for growth and have a hyper-recombination phenotype (42). This is indicative of long lived regions of ssDNA in the chromosome and is symptomatic of a defect in Okazaki fragment processing. This result suggests that a backup pathway which still involves FEN1/
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FIG. 3. Repair of adduct-damaged DNA by the FEN1/RTH1 nuclease. FEN1/RTH1 could participate in the repair of damaged DNA by the mechanism shown here. A DNA helicase displaces the damaged strand. If the damaged or adducted nucleotide can pass through the archway, FEN1/ RTH1 tracks over the damage and cleaves an oligomer containing the damaged site.
two proteins in the reaction, proving that their interaction created the stimulation. These two proteins can interact in the absence of substrate, as indicated by the binding of FEN1/RTH1 to PCNAcontaining affinity resin. Stimulation requires a large excess of PCNA over substrate molecules, presumably because PCNA freely slides on and off of the linear template. When PCNA was loaded using RFC and ATP onto M13 DNA having two adjacent primers, PCNA stimulated FEN1/RTH1 nuclease-directed cleavage of the terminal 5-nucleotide of the downstream primer (65). A PCNA mutant protein (pcna-52), incapable of trimerization, was inactive (65). The results show that PCNA only stimulates FEN1/RTH1 after encircling the substrate. Blocking the unannealed strand with a primer prevents FEN1/RTH1 nuclease-directed cleavage even in the presence of PCNA (45). This shows that PCNA does not forgive FEN1/RTH1 of its obligation to slide down the unannealed tail. Overall these results suggest that PCNA acts downstream of the displaced tail and possibly stabilizes FEN1/RTH1 at the position of cleavage. If PCNA is always loaded directionally by RFC, one face of PCNA may be designed to interact with polymerases while the other binds FEN1/RTH1. This implies that there are two PCNAs at the replication fork, one serving as the sliding clamp for DNA pol and the other interacting with FEN1/RTH1. These interactions are part of the model of the proposed pathways for Okazaki fragment processing as shown in Figs. 1 and 2. Why Does FEN1/RTH1 Nuclease Have Such a Unique Sliding Mechanism? The ssDNA sliding mechanism exhibited by the FEN1/RTH1 nuclease may have evolved because the nuclease has a dual function in DNA replication and repair. The null mutant of FEN1/ RTH1 in S. cerevisiae has an increased sensitivity to methyl methanesulfonate, an alkylating agent, implicating FEN1/RTH1 in removal of adduct-damaged nucleotides (42). The sliding mechanism of FEN1/RTH1 suggests a means by which it could participate in repair of adduct-damaged DNA. It could slide past damage on an unannealed 5-tail and then cleave the tail removing the damaged nucleotides (Fig. 3). This approach allows cleavage of a variety of types of damage, without needing to specifically recognize the structure of the damaged nucleotide. To assess the mechanism of nuclease tracking, and its ability to cleave modified DNA, adducts were placed at various locations on the tails of substrates (66). Footprint analysis using micrococcal nuclease indicates that after tracking, but before cleavage, FEN1/RTH1 protects a region of the tail 25 nucleotides long, adjacent to the cleavage site. When cis-diamminedichloroplatinum(II) (CDDP) adducts were placed within or beyond the region protected by FEN1/ RTH1, the 5-tail was cleaved. A CDDP adduct bound to the last two nucleotides at the very 5-end of an eight-nucleotide-long tail was also cleaved by FEN1/RTH1. The nuclease also removes tails
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containing adducts on the 2-position of the ribose. In contrast, a CDDP adduct just adjacent to the expected cleavage point was inhibitory to the nuclease. As discussed earlier, biotin adducts can be traversed but not if they are conjugated with streptavidin (46). This suggests an ultimate limit on adduct size, possibly imposed by the size and flexibility of the anticipated arch structure in the nuclease. It appears that the nuclease is designed to tolerate a variety of adduct structures. However, damage could inhibit catalysis if it is too close to the point of cleavage (66). This problem could be remedied in vivo by further strand displacement. DNA having abasic lesions is cleaved in a similar fashion. Abasic sites on chromosomal DNA are sensitive to type II abasic endonucleases. These cleave upstream of the damage to create a strand with a phosphorylated abasic sugar at its 5-end, annealed to a template just downstream of a second primer. FEN1/RTH1 cannot remove the abasic sugar exonucleolytically. However, strand displacement synthesis from the upstream primer can create a tail, over which the nuclease can slide, and then remove the abasic deoxyribose as part of an endonucleolytically cleaved oligomer (67). Why Are There Three Nuclear DNA Polymerases? Genetic studies in yeast demonstrate that DNA polymerases , , and are all essential (68 70). These determinations prompted suggestions that all three performed specific functions at the replication fork (4, 71, 72). DNA pol , although capable of highly processive DNA synthesis, was stimulated at high salt concentrations by interaction with PCNA (72). This suggests that it also participates in polymerase switching, having a similar role to that of DNA pol . Consistent with these observations, DNA pol was proposed to perform the majority of elongation of either the leading or lagging strand. However, reconstitution of SV40 DNA replication with purified proteins showed that both leading and lagging strand DNA replication is performed efficiently with only DNA pol and . Recent cross-linking experiments were done to determine the DNA polymerases bound to nascent DNA during replication in cell extracts (73). For SV40 replication, DNA pol but not was found to cross-link. For cellular DNA, both were found to cross-link, but mitogenic stimulation induced pol to a considerably lesser extent than DNA polymerases and . The authors concluded that only DNA pol and participate in SV40 DNA replication. Measurements with chromosomal DNA suggest that DNA pol is not participating as a sole elongating enzyme for either the leading or lagging strand. Instead it is likely to be performing an essential process that occurs during DNA replication. Recent years have seen a major expansion of our knowledge of the eukaryotic replication fork. As summarized here, nearly all of the necessary reactions and enzymes involved have been identified, characterized genetically, and reconstituted in vitro. Nevertheless, the exact complement of components, their contacts and interactions, and structure of the complex remain to be determined.
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