Flexibility of Eukaryotic Okazaki Fragment Maturation through Regulated Strand Displacement Synthesis
2008; Elsevier BV; Volume: 283; Issue: 49 Linguagem: Inglês
10.1074/jbc.m806668200
ISSN1083-351X
AutoresCarrie M. Stith, Joan F. Sterling, Michael A. Resnick, Dmitry A. Gordenin, Peter Burgers,
Tópico(s)RNA modifications and cancer
ResumoOkazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase ; (Pol ;) with 5·-flap cutting by FEN1RAD27 endonuclease. Excessive strand displacement is normally prevented by the 3·-exonuclease activity of Pol ;. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol ;) or PIF1 helicase genes can suppress lethality or growth defects of rad27Δ pol3-D520V mutants (defective for FEN1RAD27 and the 3·-exonuclease of Pol ;) that produce long flaps and of dna2Δ mutants that are defective in cutting long flaps. On the contrary, pol32Δ or pif1Δ caused lethality of rad27Δ exo1Δ double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1RAD27 and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol ; is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1RAD27 during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol ; to FEN1RAD27. Finally, RNA-DNA hybrids are more readily displaced by Pol ; than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation. Okazaki fragment maturation to produce continuous lagging strands in eukaryotic cells requires precise coordination of strand displacement synthesis by DNA polymerase ; (Pol ;) with 5·-flap cutting by FEN1RAD27 endonuclease. Excessive strand displacement is normally prevented by the 3·-exonuclease activity of Pol ;. This core maturation machinery can be assisted by Dna2 nuclease/helicase that processes long flaps. Our genetic studies show that deletion of the POL32 (third subunit of Pol ;) or PIF1 helicase genes can suppress lethality or growth defects of rad27Δ pol3-D520V mutants (defective for FEN1RAD27 and the 3·-exonuclease of Pol ;) that produce long flaps and of dna2Δ mutants that are defective in cutting long flaps. On the contrary, pol32Δ or pif1Δ caused lethality of rad27Δ exo1Δ double mutants, suggesting that Pol32 and Pif1 are required to generate longer flaps that can be processed by Dna2 in the absence of the short flap processing activities of FEN1RAD27 and Exo1. The genetic analysis reveals a remarkable flexibility of the Okazaki maturation machinery and is in accord with our biochemical analysis. In vitro, the generation of short flaps by Pol ; is not affected by the presence of Pol32; however, longer flaps only accumulate when Pol32 is present. The presence of FEN1RAD27 during strand displacement synthesis curtails displacement in favor of flap cutting, thus suggesting an active hand-off mechanism from Pol ; to FEN1RAD27. Finally, RNA-DNA hybrids are more readily displaced by Pol ; than DNA hybrids, thereby favoring degradation of initiator RNA during Okazaki maturation. The process of DNA replication in eukaryotic cells leads to the generation of a vast number of Okazaki fragments on the lagging strand of the replication fork. Approximately 50,000,000 Okazaki fragments are synthesized when a human cell replicates, and all of these need to be efficiently and accurately matured into continuous lagging strands to ensure genome integrity. Various DNA structures are generated during the synthesis and maturation of Okazaki fragments. These structures constitute the largest pool for potential DNA damage in the cell. Incomplete or poorly processed Okazaki fragments can lead to repeat expansion mutations, small duplication mutations, and to the generation of double-stranded DNA breaks (1Gordenin D.A. Kunkel T.A. Resnick M.A. Nat. Genet. 1997; 16: 116-118Crossref PubMed Scopus (189) Google Scholar). A large number of activities have been implicated in lagging strand DNA maturation. In the budding yeast Saccharomyces cerevisiae, the RAD27 gene product is the 5′-flap endonuclease FEN1. FEN1RAD27 has been assigned a dominant role in creating ligatable nicks during Okazaki maturation (reviewed in Refs. 2Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. 2004; 73: 589-615Crossref PubMed Scopus (306) Google Scholar and 3Garg P. Burgers P.M. Cell Cycle. 2005; 4: 221-224Crossref PubMed Scopus (16) Google Scholar). Strong support for the importance of RAD27 in Okazaki maturation was initially provided by the dramatic increase of small duplications up to ∼100 nt 4The abbreviations used are: nt, nucleotide(s); Pol ;, DNA polymerase ;; Pol ;-DV, Pol ; with Pol3-D520V; Pol ;* and Pol ;-DV*, two-subunit enzymes lacking Pol32; Pol, polymerase function of Pol ;; Exo, 3′-exonuclease function of Pol ;; RFC, replication factor C; RPA, replication protein A; PCNA, proliferating cell nuclear antigen. in length flanked by short repeats in rad27-null mutants (4Tishkoff D.X. Filosi N. Gaida G.M. Kolodner R.D. Cell. 1997; 88: 253-263Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). This unusual class of duplication mutations was proposed to result through ligation of an unremoved flap with the 3′-end of the downstream Okazaki fragment. This type of duplications is caused not only by the lack of RAD27 but also by a subtle rad27-p defect that severely decreases FEN1RAD27 binding to the replication clamp proliferating cell nuclear antigen (PCNA) in vitro (5Jin Y.H. Obert R. Burgers P.M. Kunkel T.A. Resnick M.A. Gordenin D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5122-5127Crossref PubMed Scopus (130) Google Scholar). Consistent with these genetic observations, biochemical studies showed that FEN1RAD27 is ideally suited to create ligatable nicks from 5′-flaps generated by the lagging strand DNA polymerase ; (6Maga G. Villani G. Tillement V. Stucki M. Locatelli G.A. Frouin I. Spadari S. Hubscher U. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14298-14303Crossref PubMed Scopus (112) Google Scholar, 7Ayyagari R. Gomes X.V. Gordenin D.A. Burgers P.M. J. Biol. Chem. 2003; 278: 1618-1625Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Based on these biochemical and genetic insights, we and others previously proposed that FEN1RAD27 is part of a minimal core Okazaki maturation machinery that efficiently processes the vast majority of the Okazaki fragments in the cell and can be assisted by a number of auxiliary factors that function under specific circumstances when the core machinery falters (Fig. 1) (reviewed in Ref. 8Garg P. Burgers P. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 115-128Crossref PubMed Scopus (215) Google Scholar). The core maturation machinery consists of three factors: Pol ;, FEN1RAD27 and DNA ligase I. These three factors are held in a complex through interactions with PCNA. FEN1RAD27 and DNA ligase I are both single-subunit enzymes with well characterized biochemical activities (reviewed in Refs. 2Liu Y. Kao H.I. Bambara R.A. Annu. Rev. Biochem. 2004; 73: 589-615Crossref PubMed Scopus (306) Google Scholar and 9Tomkinson A.E. Vijayakumar S. Pascal J.M. Ellenberger T. Chem. Rev. 2006; 106: 687-699Crossref PubMed Scopus (212) Google Scholar). S. cerevisiae Pol ; consists of three subunits: Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa). The POL3 and POL31 genes are essential for yeast viability. POL32 deletion mutants are viable, but show defects in DNA replication, DNA recombination, and repair and in mutagenesis (10Gerik K.J. Li X. Pautz A. Burgers P.M. J. Biol. Chem. 1998; 273: 19747-19755Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 11Huang M.E. Rio A.G. Galibert M.D. Galibert F. Genetics. 2002; 160: 1409-1422Crossref PubMed Google Scholar, 12Johansson E. Garg P. Burgers P.M. J. Biol. Chem. 2004; 279: 1907-1915Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Gibbs P.E. McDonald J. Woodgate R. Lawrence C.W. Genetics. 2005; 169: 575-582Crossref PubMed Scopus (159) Google Scholar, 14Lydeard J.R. Jain S. Yamaguchi M. Haber J.E. Nature. 2007; 448: 820-823Crossref PubMed Scopus (376) Google Scholar). These three subunits of Pol ; are conserved in all organisms; however, the enzyme from Schizosaccharomyces pombe and from higher eukaryotic organisms contains an additional small fourth subunit that increases the stability of the enzyme (15Podust V.N. Chang L.S. Ott R. Dianov G.L. Fanning E. J. Biol. Chem. 2002; 277: 3894-3901Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Biochemical studies of the S. cerevisiae three-subunit Pol ; compared with the two-subunit Pol3-Pol31 enzyme lacking the Pol32 subunit, designated as Pol ;*, indicate that the latter enzyme has a decreased processivity of DNA synthesis and a decreased interaction with PCNA (16Burgers P.M. Gerik K.J. J. Biol. Chem. 1998; 273: 19756-19762Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Biochemical and genetic studies indicate that Pol32 is also important for physical interactions of Pol ; with Pol ;-primase, suggesting that Pol32 may coordinate enzymatic activities during lagging strand synthesis and Okazaki maturation (12Johansson E. Garg P. Burgers P.M. J. Biol. Chem. 2004; 279: 1907-1915Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 17Huang M.E. Le Douarin B. Henry C. Galibert F. Mol. Gen. Genet. 1999; 260: 541-550Crossref PubMed Scopus (31) Google Scholar). We previously noted that defects in the 3′-5′-exonuclease activity of Pol ; can be lethal when combined with rad27Δ or even with minor defects in RAD27 (5Jin Y.H. Obert R. Burgers P.M. Kunkel T.A. Resnick M.A. Gordenin D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5122-5127Crossref PubMed Scopus (130) Google Scholar). The same combined lethality was observed for POL3 mutants that, although fully proficient for exonuclease activity, show impaired switching from the polymerase to the exonuclease domain (18Jin Y.H. Garg P. Stith C.M. Al Refai H. Sterling J. Weston L. Kunkel T. Resnick M.A. Burgers P.M. Gordenin D.A. Mol. Cell. Biol. 2005; 25: 461-471Crossref PubMed Scopus (60) Google Scholar). Importantly, double mutant combinations that are not lethal show an increased rate of small duplication mutations, a hallmark signature of defects in lagging strand DNA replication. This led us to propose that strand displacement synthesis by the polymerase (Pol) activity of Pol ; is countered by the competing 3′-5′-exonuclease (Exo) activity of the enzyme, also contained within the catalytic Pol3 subunit of polymerase ;. In agreement with this proposal, our in vitro studies showed that the functional interaction between the three enzymatic activities (i.e. polymerase and 3′-exonuclease activities of Pol ; and the 5′-flap endonuclease activity of FEN1) are important for efficient maturation of Okazaki fragments (19Jin Y.H. Ayyagari R. Resnick M.A. Gordenin D.A. Burgers P.M. J. Biol. Chem. 2003; 278: 1626-1633Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). When Pol ; reaches the 5′-end of the downstream Okazaki fragment, it continues an additional 1–2-nt synthesis in a strand displacement mode (20Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (175) Google Scholar). FEN1 removes the small 5′-flap and creates a ligatable nick for DNA ligase on the border between adjacent fragments, provided that no RNA is left on the downstream Okazaki fragment. Otherwise, the RNA primer is removed by iterative processing through alternate Pol ; and FEN1 action, called nick translation, where a single ribonucleotide is typically removed with each cycle (Fig. 1). This forward movement by Pol ; can be counteracted by its Exo activity. The nuclease activity of Pol ; is generally considered to be a proofreading function to assure high fidelity DNA replication (21Simon M. Giot L. Faye G. EMBO J. 1991; 10: 2165-2170Crossref PubMed Scopus (183) Google Scholar). However, the nuclease activity also plays a crucial role in Okazaki fragment maturation (18Jin Y.H. Garg P. Stith C.M. Al Refai H. Sterling J. Weston L. Kunkel T. Resnick M.A. Burgers P.M. Gordenin D.A. Mol. Cell. Biol. 2005; 25: 461-471Crossref PubMed Scopus (60) Google Scholar). Exo-mediated 3′-degradation increases opportunities for generating and maintaining ligatable nicks. In fact, Pol ; has the remarkable ability to idle at a nick. Idling is the iterative process of limited (∼1–2-nt) strand displacement synthesis at the nick, followed by switching to the Exo domain and degradation of the displacing strand until the nick position has been reached again. In this fashion, Pol ; not only has the ability to resolve small 5′-flaps by degradation of the displacing 3′-strand (Fig. 1) but also maintains a dynamic relationship with the nick position. This idling capacity at a nick is unique to the lagging strand Pol ;, and it is not displayed by leading strand Pol ? (20Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (175) Google Scholar). This efficient minimal core machinery consisting of Pol ; and FEN1 can be augmented by several auxiliary proteins in order to mature Okazaki fragments that might be difficult to process because of DNA structural problems or if there is an accumulation of long flaps because of lack of flap cutting by FEN1. Among these is Dna2, which has both 3′-5′-helicase and nuclease activity. Its nuclease activity is essential for viability (22Lee K.H. Kim D.W. Bae S.H. Kim J.A. Ryu G.H. Kwon Y.N. Kim K.A. Koo H.S. Seo Y.S. Nucleic Acids Res. 2000; 28: 2873-2881Crossref PubMed Scopus (71) Google Scholar, 23Budd M.E. Choe W. Campbell J.L. J. Biol. Chem. 2000; 275: 16518-16529Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Pif1 is a 5′-3′-helicase that functions in mitochondrial DNA maintenance and in telomere homeostasis (24Boule J.B. Zakian V.A. Nucleic Acids Res. 2006; 34: 4147-4153Crossref PubMed Scopus (104) Google Scholar). It was also suggested to counteract formation of ligatable nicks by creating larger flaps. This conclusion was drawn because deletion of PIF1 rescues the lethality of dna2 mutants (25Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar). Recent biochemical studies have shown that the presence of Pif1 during gap filling by Pol ; favors the generation of longer flaps, thereby providing a biochemical rationale for an essential Dna2 requirement in cells carrying Pif1 (26Rossi M.L. Pike J.E. Wang W. Burgers P.M. Campbell J.L. Bambara R.A. J. Biol. Chem. 2008; 283: 27483-27493Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Other factors that may act during lagging strand replication include the flap exoendonuclease Exo1, RNase H2, and the RecQ-like helicase Sgs1 (27Chen J.Z. Qiu J. Shen B. Holmquist G.P. Nucleic Acids Res. 2000; 28: 3649-3656Crossref PubMed Scopus (27) Google Scholar, 28Tran P.T. Erdeniz N. Dudley S. Liskay R.M. DNA Repair (Amst.). 2002; 1: 895-912Crossref PubMed Scopus (100) Google Scholar, 29Ii M. Brill S.J. Curr. Genet. 2005; 48: 213-225Crossref PubMed Scopus (34) Google Scholar). Interestingly, neither exo1Δ nor rnh2Δ (eliminating RNase H2) mutants cause small duplications. It is not clear under what conditions these auxiliary Okazaki maturation factors are called into action. One prevailing model is that when flaps have grown to excessive length, they are no longer substrates for FEN1; nor can they be restored to a nick structure by the Exo activity of Pol ;. Long 5′-flaps, perhaps longer than 25 nt, to which DNA-binding proteins are bound (e.g. the single-stranded binding protein RPA) fail to engage FEN1 (reviewed in Ref. 30Kao H.I. Veeraraghavan J. Polaczek P. Campbell J.L. Bambara R.A. J. Biol. Chem. 2004; 279: 15014-15024Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), whereas backup by the Exo activity of Pol ; is also limited to flaps that are only a few nt long (20Garg P. Stith C.M. Sabouri N. Johansson E. Burgers P.M. Genes Dev. 2004; 18: 2764-2773Crossref PubMed Scopus (175) Google Scholar). Apparently, efficient and faithful Okazaki fragment maturation requires a proper balance between DNA synthesis, strand displacement synthesis, and the capability for 5′- and 3′-degradation, as indicated in the model in Fig. 1. One prediction of the model is that under conditions where strand displacement synthesis is reduced, there would be less of a need for nuclease degradation of long flaps. Genetic experiments with mutants of the auxiliary Okazaki maturation nuclease DNA2 supported this view in that the temperature sensitivity of conditional dna2 mutants could be suppressed by deletion of the POL32 gene (25Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar). It was suggested that the suppressive effect of the POL32-null mutation was caused by a reduction in strand displacement synthesis by the two-subunit form of Pol ;, resulting in the creation of a smaller number of the infrequent substrates that would require Dna2 for processing. Although consistent with the data, this hypothesis lacked experimental verification. We have undertaken comprehensive genetic and biochemical studies to understand the role that Pol32 plays in the core machinery required for Okazaki maturation. We found that the distribution of flap sizes is determined by several competing activities in vitro. The length of the flap generated during a processive cycle of strand displacement synthesis by Pol ; is one indicator of the length of the flap to be cut by FEN1. A second indicator is the capacity for 3′-degradation by the Exo activity of Pol ;. Third, the presence of FEN1 curtails processive strand invasion synthesis by Pol ; in favor of flap cutting. Finally, in keeping with its function in Okazaki fragment maturation, Pol ; shows an increased capacity to displace RNA-DNA hybrids. If Pol ; lacks Pol32 (designated Pol ;*), there is decreased processivity of strand displacement as well as reduced 3′-degradation. As a consequence, the size distribution of short flaps (<10 nt) is not affected by Pol32. However, long flaps are only detected when Pol32 is present. These in vitro findings in parallel with our genetic studies provide a compelling explanation of how cells deal with the balance between flap generation and cutting. Importantly, the combination of the exonuclease activity of Pol ; and its third subunit Pol32 provide flexibility to the eukaryotic Okazaki maturation machinery. Yeast Strains—All strains were derived from ALE100, ALE101, ALE1000, and ALE1001, isogenic to CG379 (MAT; ade5-1 his 7-2 leu 2-3, 112trp1-289 ura3Δ). In addition, strains carried alleles of LYS2 in two chromosomal locations (see Ref. 5Jin Y.H. Obert R. Burgers P.M. Kunkel T.A. Resnick M.A. Gordenin D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5122-5127Crossref PubMed Scopus (130) Google Scholar and references therein). Completely homozygous isogenic diploid strains were obtained after transformation of the haploid wild type strain with a YEpHO-URA3 plasmid carrying the URA3 selectable marker and HO-endonuclease gene under control of its own promoter. HO-endonuclease causes mating type switching within the population of transformed cells that allows mating and diploid formation. Single colony isolates of diploids that had lost the HO plasmid were taken. Isogenic diploids homozygous for pol3-L523H/pol3-L523H were created by crossing two isogenic isolates of MATa pol3-L523H and MAT; pol3-L523H. Diploids with single and multiple heterozygosities for deletion alleles were obtained by transforming homozygous wild type diploids with deletion cassettes carrying selectable markers. The RAD27 gene was replaced with the RAD27: URA3-Blast cassette (31Sommers C.H. Miller E.J. Dujon B. Prakash S. Prakash L. J. Biol. Chem. 1995; 270: 4193-4196Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Other deletions were obtained by inserting antibiotic-resistant cassettes (32Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1389) Google Scholar). Multiple deletions within the same heterozygous diploid strain were introduced separately by successive transformations. Isogenic diploid strains carrying the triple heterozygous mutations POL3/pol3-5DV RAD27/rad27Δ POL32/pol32Δ were obtained by crossing isogenic isolates of the MAT; pol3-5DV (note that pol3-5DV was also referred as pol3-D520V in Ref. 18Jin Y.H. Garg P. Stith C.M. Al Refai H. Sterling J. Weston L. Kunkel T. Resnick M.A. Burgers P.M. Gordenin D.A. Mol. Cell. Biol. 2005; 25: 461-471Crossref PubMed Scopus (60) Google Scholar) with isogenic isolates of the MATa rad27Δ pol32Δ genotype. All crosses and sporulations were carried out at least in duplicate with independent isolates of the relevant strains. Tetrad Analysis—Effects of eliminating Pol32 or Pif1 on the viability of single and multiple mutants presumably defective in Okazaki maturation were studied in the immediate meiotic progeny of heterozygous diploids. This allowed us to minimize effects due to the accumulation of suppressor mutations that could be selected if multiple mutants were obtained by consecutive transformations of a haploid strain in order to disrupt the genes of interest. Asci of double or triple heterozygous diploids were dissected on YPDA plates and incubated at 23 or 30 °C. In order to allow spores carrying slow growing genotypes to form colonies, plates containing very small colonies were incubated up to 7–10 days. Colonies were then replica-plated onto both YPDA and diagnostic media to identify mating type and gene deletions by the presence of markers. YPDA plates were incubated at 23, 30, and 37 °C to identify possible temperature sensitivities of single and multiple mutants. The presence of the pol3-5DV mutation was determined by diagnostic restriction digestion of a PCR product. We explored genetic interactions throughout this paper in the progeny of multiple heterozygous diploids, where all possible combinations of mutant alleles are represented by colonies that grew from a fresh meiotic progeny. Replication Proteins—Overexpression and purification of Pol ; and Pol ;-DV (DNA polymerase ; with pol3-5DV mutation) from yeast has been described in detail (33Fortune J.M. Stith C.M. Kissling G.E. Burgers P.M. Kunkel T.A. Nucleic Acids Res. 2006; 34: 4335-4341Crossref PubMed Scopus (60) Google Scholar). In order to overproduce Pol ;* and Pol ;-DV*, the overexpression strain contained plasmids pBL335 (GAL-GST-POL3) or pBL335-DV (GAL-GST-pol3-D520V), respectively, together with plasmid pBL338 (GAL-POL31 (16Burgers P.M. Gerik K.J. J. Biol. Chem. 1998; 273: 19756-19762Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar)). After growth, galactose induction, cell lysis, and glutathione affinity chromatography, the preparation was subjected to 3C-protease treatment to remove the glutathione S-transferase purification tag, followed by MonoS chromatography, as described for Pol ; (33Fortune J.M. Stith C.M. Kissling G.E. Burgers P.M. Kunkel T.A. Nucleic Acids Res. 2006; 34: 4335-4341Crossref PubMed Scopus (60) Google Scholar). Replication protein A (RPA), PCNA, Replication factor C (RFC), and FEN1 were purified from Escherichia coli overproduction strains (7Ayyagari R. Gomes X.V. Gordenin D.A. Burgers P.M. J. Biol. Chem. 2003; 278: 1618-1625Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 34Henricksen L.A. Umbricht C.B. Wold M.S. J. Biol. Chem. 1994; 269: 11121-11132Abstract Full Text PDF PubMed Google Scholar, 35Gomes X.V. Gary S.L. Burgers P.M. J. Biol. Chem. 2000; 275: 14541-14549Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). All other enzymes were obtained commercially. DNA Substrates—All oligonucleotides were obtained from Integrated DNA Technologies and purified by polyacrylamide gel electrophoresis or high pressure liquid chromatography before use. They are as follows: Bio-V6, 3′-biotin-T31CCCTTCCCTCTCCCTCCTCTTCTTCCCTCT25CCAAGGTGGTTTGTTTTGGTTGGGTTGA-biotin-5′; C12, 5′-AGGGAAGGGAGAGGGAGGAGAAGAAGGGAG; C6, 5′-GGTTCCACCAAACAAAACCAACCCAAC; RC6, 5′-GGUUCCACCAAACAAAACCAACCCAAC (ribonucleotides underlined). The 113-nt 5′ and 3′ biotinylated template Bio-V6 was prepared by hybridizing two half-oligonucleotides to a bridging primer followed by ligation with T4 DNA ligase and purification by preparative urea-PAGE (see Fig. 3A). For strand displacement assays, the primer oligonucleotide C12 was 5′-32P-labeled, and 5 pmol of 32P-C12 was hybridized to 7.5 pmol of Bio-V6 and 15 pmol of pC6 or pRC6 (made by phosphorylation of C6 and RC6 with cold ATP). The oligonucleotides were hybridized to the template at 70 °C. For nick translation assays, either primer C6 or RC6 was 5′-32P-labeled, and 5 pmol of the labeled oligonucleotide was hybridized to 7.5 pmol of Bio-V6 together with 15 pmol of C12. For idling assays, 5 pmol of Bio-V6 was hybridized with 7.5 pmol of C12 and 7.5 pmol of pC6. After hybridization, streptavidin was added in a 2-fold molar excess to all template-primer substrates. Strand Displacement and Nick Translation Assays—In all assays in this study, the DNA concentration was 7 nm. Standard 20-;l assays contained 20 mm Tris-HCl, pH 7.8, 1 mm dithiothreitol, 100 ;g/ml bovine serum albumin, 8 mm MgAc2, 0.5 mm ATP, 110 mm NaCl, 100 ;m each dNTP, 140 fmol of DNA substrate (see above and in the figures), 1 pmol of RPA, 200 fmol of RFC, and 400 fmol of PCNA. The DNA was preincubated with RPA, PCNA, and RFC for 1 min at 30 °C, and the reaction was started by adding the relevant DNA polymerase at the indicated concentrations. Added NaCl was adjusted such that the final concentration, including additions from enzyme storage buffers, was 110 mm. In nick translation assays, FEN1 (200 fmol) was added together with the indicated polymerase. Aliquots were quenched in 20 mm EDTA, 0.4% SDS (final) and heated at 50 °C for 15 min. After the addition of 95% formamide to a 60% final concentration, the samples were heated at 95 °C for 2 min and analyzed on a 12% (strand displacement) or 20% (nick translation) denaturing polyacrylamide gel. The gels were dried and subjected to PhosphorImager analysis. In order to allow accurate quantitation of weak signals, the gels were exposed for several hours and for 2 days. PhosphorImager file intensities were correlated by quantitation of intermediate signals that were in the linear response range for both exposures. Quantification was carried out using ImageQuant software (GE Healthcare). The images in the figures were contrast-enhanced for visualization purposes. Idling Assays—The above assay was scaled up to 50 ;l. It contained unlabeled DNA, 10 ;m [;-32P]dGTP, and a 100 ;m concentration of the other dNTPs and reaction conditions and enzymes as indicated above and in the legend to Fig. 4. Aliquots of 10 ;l were taken after various times, from 1 to 7 min, quenched by the addition of 5 ;l of stop buffer (25 mm EDTA, 1% SDS, and a 5 mm concentration each of the relevant nonradioactive dNTP and dNMP). Reaction products were analyzed by thin layer chromatography on polyethyleneimine/cellulose in 0.5 m LiCl/HCOOH, followed by PhosphorImager analysis. Idling rates were calculated from the linear slopes of time courses. Previous genetic studies have established several networks of interactive factors involved in Okazaki fragment maturation. One network links the nuclease activities of FEN1RAD27 and Dna2, showing that these factors have compensatory activities. Overexpression of DNA2 suppresses the temperature sensitivity of rad27Δ, and overexpression of RAD27 suppresses the temperature sensitivity of a dna2-1 mutant (36Budd M.E. Campbell J.L. Mol. Cell. Biol. 1997; 17: 2136-2142Crossref PubMed Scopus (193) Google Scholar). A second network links Dna2 with the helicase Pif1 and the Pol32 subunit of Pol ;. Pif1Δ suppresses the lethality of the dna2Δ mutant, and the additional deletion of POL32 improves growth in the triple mutant (25Budd M.E. Reis C.C. Smith S. Myung K. Campbell J.L. Mol. Cell. Biol. 2006; 26: 2490-2500Crossref PubMed Scopus (167) Google Scholar). A third genetic network includes the core activities for Okazaki maturation. It centers around the nuclease activities of Pol ; and FEN1RAD27, revealing a synthetic lethality or showing a signature hypermutability of small duplications when even mild mutations in POL3 and RAD27 are combined (18Jin Y.H. Garg P. Stith C.M. Al Refai H. Sterling J. Weston L. Kunkel T. Resnick M.A. Burgers P.M. Gordenin D.A. Mol. Cell. Biol. 2005; 25: 461-471Crossref PubMed Scopus (60) Google Scholar). In order to gain additional insights into this core network, we have made a series of multiple mutants that probe the interactions and interdependencies of these networks. We analyzed a large number of spores in the progeny of multiple heterozygous diploids to identify all possible viable single and multiple mutant genotypes based on their associated selection markers or by PCR/restriction digest genotyping. This approach reduces the propagation of potentially unstable mutants to a practically achievable minimum and therefore limits the possibility of generating suppressor mutations of poorly growing mutants. This important technical imposition allowed us to draw conclusions about genetic interactions that are not affected by the generation of suppressor mutants during prolonged growth. We based our experimental design on the use of two genetic defects (pol32Δ and pif1Δ) that may lead to reduced flap formation via different mechanisms. Pol
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