Proc. Nate Acad. Sci. USA Vol. 79, pp. 7253-7257, December 1982 Biochemistry

Deletion mutants defining the replication factor Y effector site sequences in pBR322 DNA (ATPase/origin function) W. C. SOELLER AND K. J. MARIANS Department of Developmental Biology and Cancer, Division ofBiological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461 Communicated byjerard Hurwitz, September 15, 1982 ABSTRACT The Escherichia coli DNA replication factor Y, which are on opposite strands, have been localized to nucleo- along with other genetically undefined replication proteins, is in- tides 2,114-2,185 and 2,353-2,416 for the H and L strand volved in a dnaB-, dnaC-, and dnG-dependent pathwayofprimer sites,* respectively (14). Shlomai and Kornberg (15) have shown formation on 4X174 single-stranded circular DNA. In addition, that the 4X174 Y site can form a structure resistant to E. coli replication factor Y has a site-specific, single-stranded DNA-de- exonuclease VII, an enzyme that degrades ss DNA from either pendent ATPase activity. We have previously demonstrated the end but leaves duplex DNA intact. Subsequently, we have presence of two factor Y effector sites on pBR322 DNA. When shown that, although the pBR322 H strand site can also form inserted into the filamentous phage fIR229, these sites can func- a resistant tion as rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent structure to exonuclease VII, the pBR322 L strand origins of DNA replication. We report here the construction of site cannot (14). deletion mutants of the two pBR322 factor Y effector sites. These Insertion of small restriction fragments containing the deleted sites no longer function as effectors for factor Y ATPase pBR322 Y sites into fl DNA conferred upon the recombinants activity nor as templates for rifampicin-resistant dnaB-, dnaC-, the ability to be converted to replicative form (RF) DNA via a and dnaG-dependent DNA synthesis. We conclude that the DNA rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent path- sequences required for factor Y ATPase activity and origin func- way in vitro (16). This suggested that the Y sites were capable tion are likely to be identical. ofacting as origins ofreplication because their presence allowed contiguous nonreplicating DNA sequences to be duplicated in Studies on the initiation of DNA replication using small single- a specific manner. stranded (ss) DNA bacteriophage genomes as templates have Factor Y is presumably the key to the assembly of the pri- identified three basic enzymatic processes of de novo primer mosome. Thus, acomplete understanding ofits interaction with formation in Escherichia coli. (i) Unique regions of the bacte- its effector sites will be important in the understanding of the riophage M13 and fl viral strand , when coated with the initiation of lagging-strand DNA synthesis. To reach such an E. coli ss DNA binding protein (ssb), direct the formation of a understanding we have undertaken the study ofthe phenotype short RNA primer by RNA which is subsequently ofmutant factor Y sites constructed in vitro. We report here the extended by the E. coli DNA polymerase III elongation system construction oftwo mutant factor Y sites containing small dele- (1-4). (ii) DNA sequences within the intercistronic region of tions in each ofthe pBR322 factor Y effector sequences. These bacteriophage G4 viral DNA can act as the unique site for deleted sites can no longer function as effectors for the factor primer formation by the E. coli dnaG protein () when Y ATPase activity nor as templates for rifampicin-resistant the DNA is coated with ssb (5-7). (iii) Priming of 4X174 viral dnaB-, dnaC-, and dnaG-dependent DNA synthesis. This coin- strands requires the formation ofapre-priming complex termed activation ofthe two activities associated with factor Y indicates "the " (8), consisting ofthe dnaB, dnaC, and. dnaG that the DNA sequences required for ATPase effector activity gene products and the genetically undefined proteins replica- and origin function are likely to be identical. tion factors X, Y, and Z (9) (i, n, n', and n") (10). The primosome has been shown to move along the template DNA strand in a MATERIALS AND METHODS 5' -- 3' direction, generating primers at multiple sites along Bacterial Strains and Phages. E. coli strains and their rel- the genome (8, 11, 12). This dnaB-, dnaC-, and dnaG-depen- evant genotypes were K38(Hfr C), BT1029 (polA, dnaB), PC22 dent pathway ofprimer formation is considered to be a model (polA, dnaC), NY73 (polA, dnaG), and HMS83 (polA, polB) (see for the multiple priming required for lagging-strand DNA syn- ref. 16 for complete genotypes). SY562 (pJK129) (17) was a gift thesis of the E. coli . fromJ. Kobori and A. Kornberg (Stanford University). RLM 782 E. coli replication factor Y (or n') is a ss DNA-dependent and RLM 757 were gifts from R. McMacken (Johns Hopkins ATPase whose activity can be induced by short, specific DNA University). Bacteriophage strains used were 45X174 am3 sequences. Five such sequences have been identified. The 55- (lysis-, gene E), flR229 (18), and recombinant phages flYB5 -long factor Y effector sequence located within the and f1YE3 which contain the H and L strand factor Y effector F-G intergenic space of 4X174 viral strand DNA presumably acts as the origin of complementary strand synthesis (11). We Abbreviations: ss, single-stranded; ss(c) DNA, ss circular DNA; RF have identified two factor Y effector sequences on the DNAs DNA, double-stranded DNA in the replicative form; Y site, single- ofthe plasmids ColEl and pBR322 near the origin ofreplication stranded DNA region functioning as an effector for E. coli DNA rep- of the plasmids (13). lication factor Y; ssb, E. coli single-stranded DNA binding protein; bp, (s). The maximal limits ofthe two pBR322 factor Y effector sites, * We refer to the pBR322 leading- and lagging-strand templates as H and L, respectively. In ColEl DNA replication, the leading-strand The publication costs ofthis article were defrayed in part by page charge template is the H strand and the lagging strand template is the L payment. This article must therefore be hereby marked "advertise- strand. The H and L designations of the two DNA strands of ColEl ment' in accordance with 18 U. S. C. §1734 solely to indicate this fact. reflect heavy and light density, respectively. 7253 Downloaded by guest on September 26, 2021 7254 Biochemistry: Soeller and Marians Proc. Natl. Acad. Sci. USA 79 (1982) sites of pBR322 DNA, respectively (16). was removed by repeated ethanol precipitation. Ligation con- Enzymes and DNAs. The restriction endonucleases EcoRI, ditions were as recommended by the manufacturer. Ligation Hha I, Msp I, HgiAI, BamHI, and Pvu II were purchased from of DNA fragments digested with either Msp I or Hha I was New England BioLabs. T4 polynucleotide kinase, T4 polynu- performed at 40C at a DNA concentration of 50 pg/ml. Liga- cleotide ligase, and bacterial alkaline phosphatase were pur- tion of DNA fragments with EcoRI cohesive ends to the EcoRI- chased from P-L Biochemicals, Bethesda Research Laborato- digested flR229 RF vector DNA was carried out at 16'C with ries, and Millipore, respectively. Factor Y was purified by a 12 pug of DNA per ml. E. coli K38 was transformed and plaque combination ofthe procedures of Wickner and Hurwitz (19) and hybridizations were performed as described (28-30). Inserts of Shlomai and Kornberg (20). The dnaB and dnaG gene products the recombinant flYB5 and f1YE3 RF DNAs were isolated and were purified from E. coli strains that overproduce them (RLM converted into 32P-labeled probes by nick-translation (31). 782 and RLM 757, respectively) by a combination of the pro- cedures of Reha-Krantz and Hurwitz (21) and Lanka et al. (22) for the dnaB protein and Benz et aL (23) for the dnaG protein. RESULTS The dnaC protein was also purified, by an unpublished pro- Construction and Isolation of Deletion Mutants of the cedure, from a strain that overproduces it (SY562). The ssb was pBR322 Factor Y Effector Sites. The construction and char- a gift from J. Chase (this institution). 4X174 RFI [3H]DNA, acterization of the recombinant phages flYB5 and f1YE3, con- 4X174 ss circular DNA [ss(c) DNA], fl, and recombinant fl RFI taining the pBR322 H strand and L strand factor Y effector sites, and ss(c) DNAs were prepared as described (24-26). respectively, have been described (16). The maximal limits of FactorY ATPase Assay. Reaction mixtures (25 1.d) contained these sites have been determined to be from pBR322 nucleotide 50 mM Tris HCl (pH 8.4 at 300C), 20 mM KCI, bovine serum residues 2,114-2,185 and 2,353-2,416 for the H and L strand albumin at 20 Ag/ml, rifampicin at 10 ,g/ml, 1.0 mM MgCl2, sites, respectively (14). Examination of a detailed restriction 0.7mM [y-32P]ATP (10-50 cpm/pmol), 1.0 mM dithiothreitol, endonuclease map of this region of pBR322 revealed the pres- 18 fmol ss DNA (as molecules), 1 ,tg of ssb per 180 pmol of ence of only two Msp I sites, 34 apart at positions nucleotide residues, and 2.4 units offactor Y (fraction 8) (1 unit 2,121 and 2,154 within the pBR322 DNA sequence present in corresponds to the release of 1 nmol ofPi in 30 min at 30°C) and flYBM DNA. Similarly, we found only two Hha I sites, 33 nu- were incubated at 30°C for 10 min. Pi formation was determined cleotides apart, at positions 2,352 and 2,385 within the pBR322 by the method of Conway and Lipmann (27). DNA sequence present in f1YE3 DNA. Fortuitously, these sites ss(c) DNA -* RF DNA Replication. Reaction mixtures (50 lay within or overlapped the previously identified factor Y ef- ,ul) contained 20mM Tris HCI (pH 7.5 at 30°C), 10 mM MgCl2, fector sequences. By digesting each respective DNA insert with 1 mM dithiothreitol, 40 ,M [a-3P]TTP (100-500 cpm/pmol), the appropriate enzyme, eliminating the central fragment span- 40 ,uM dCTP, 40 ,uM dATP, 40 ,uM dGTP, 2mM ATP, 100 pzM ningthe restriction endonuclease sites, and ligatingthe flanking UTP, 100 ,uM GTP, 100 ,uM CTP, rifampicin at 10 ,ug/ml, 10 ends of the insert back together, small deletions ofeach factor mM NAD+, and 4-8 ,l of the appropriate crude ammonium Y effector site could be generated. The details ofthe procedures sulfate receptor fraction (the protein concentrations of the re- used to construct these mutants are given in the legend to Fig. ceptor fractions prepared from BT1029, PC22, and NY73 were 1. The fragments now containing deletions were reinserted into 55, 94, and 70 mg/ml, respectively) and were incubated at 300C EcoRI-linearized flR229 RF DNA and cloned. The resulting for 15 min. Acid-insoluble radioactivity was then measured. plaques were screened by plaque hybridization (30) using as Where indicated, 0.31, 0.32, and 0.18 unit (1 unit corresponds probes the pBR322 DNA fragments contained in flYB5 and to the incorporation of 1 nmol ofTMP into acid-insoluble prod- f1YE3 RF DNAs which had been labeled with 3aP by nick-trans- uct in 30 min at 300C) of the dnaB, dnaC, and dnaG proteins, lation (31). respectively, was added. Receptor fractions were prepared by Ten plaques that were positive with the flYB5 insert probe using 45% (wt/vol) ammonium sulfate as described (16). were picked forfurther study. RFI DNAwas purified from each Techniques Used for Cloning. Restriction endonuclease isolate, the DNAs were digested with EcoRI, and the resultant digestions were performed according to conditions recom- digest was electrophoresed through a 5% polyacrylamide gel in mended by the manufacturer. DNA fragments were separated order to determine the size ofthe inserted fragment. Ofthe 10 on 5% polyacrylamide gels (30:1 acrylamide/bisacrylamide) RF DNAs so treated, 5 contained inserted fragments that mi- with 50 mM Tris/45 mM borate/i mM EDTA, pH 8.3, as the grated faster than the wild-type parental inserted fragment. electrophoresis buffer (TBE). Fragments were visualized by One isolate did contain a wild type-size fragment. Phage whose ethidium bromide staining and recovered by electroelution in RF DNAs showed inserted fragments smaller than the starting TBE buffer followed by overnight dialysis in 10 mM Tris HCI, material were plaque-purified, retested for fragment size, and pH 7.5 (at 4°C)/1.0 mM EDTA/10 mM NaCl (TEN buffer). used to purify ss(c) DNA. Twelve ofthe plaques that were pos- The dialyzed DNA solution was filtered through siliconized itive with the flYE3 insert probe were treated in a similar man- glass wool to remove residual acrylamide fragments, extracted ner. with an equal volume ofphenol followed by ether extraction to Reinsertion ofthe EcoRI fragments containing deletions into remove residual phenol, and ethanol precipitated from 0.16 M the flR229 vector DNA could occur in either orientation, pre- Tris HCl (pH 8.5 at 40C). 5'-Terminal phosphate residues were sumably at equal frequency. Thus, in each case, either the de- removed from DNA fragments by treatment with 0.1-0.2 unit leted Y sites or their complements would be contiguous with of bacterial alkaline phosphatase at 60°C for 2-3 hr in 30 mM the viral (+) strand ofthefl vector DNA. In order to determine Tris HCI, pH 8.8 (at 37C)/50 AM ZnSO4. The reaction was which isolates contained the deleted Y sites in the correct ori- terminated by extraction with phenol and then ether. The DNA entation, appropriate restriction endonuclease analysis of the fragments were recovered by ethanol precipitation. Dephos- RFI DNAs was performed (Fig. 2). phorylated DNA fragments were labeled with 32P by incubation Characterization of the Deleted Sequences. Two ofthe iso- for 60 min at 37°C with T4 polynucleotide kinase (60 units/ml) lates with the correct orientation-flYBd9, derived from in 3 uM [y-32P]ATP (2.8 x 106 cpm/pmol), 50 mM Tris HCI, flYB5, and flYEdl, derived from f1YE3-were examined in pH 7.5 (at 300C)/10 mM dithiothreitol/10 mM MgCl2 contain- detail to determine the extent and position ofthe deleted DNA. ing bovine serum albumin at 50 kkg/ml. Residual [y-32P]ATP The inserted fragments were released by EcoRI digestion and Downloaded by guest on September 26, 2021 Biochemistry: Soeller and Marians Proc. Natl Acad. Sci. USA 79 (1982) 7255

2700 2400 2100 1800 A B C D E F G H HgiA I RsaI FnuDIj HPvu Hoe 11[

/nserted andy /coned in f1R229 EcoR EcoRI EcoRI,--. EcoRI

fIYE3 fIYB5

RF I molecules restricted with EcoRI I 247 bp 341 bp -77 HhaI HhaI MspI MspI

HhaII Restriction digest I MspI

72 33 142bp 131 34 176bp 'FIG. 2. Orientation of inserted fragments in thefactor Y site dele- tion derivatives of recombinant DNAs. flYB5 (lane B), flYB1 (lane 0), and f1YBd9 (lane D) RFI DNAs were cleaved with HgiAI, BamHI, G- 50 Column andPvu IIM(see Fig. 1 for restrictionmap). f1YE3 (lane E), f1YE5 (lane F), and flYEdl (lane G) RFI DNAs were cleaved with HgiAI and 72 142bp 131 176bp BamHI. Hae Il-digested 4X174 RFI DNA (lane A) andMsp I-digested f1R229 RFI DNA (lane H) served asmarkers. The samemobility of the HgiAl-Pvu II fragment (273 bp) in lanes B and D is indicative of the identical orientation of the inserts in fIYB5 and f1YBd9, in contrast Ligation to flYB1 which contains the insert in the opposite orientation (lane C). The difference in size of the Pvu ll-BamHI fragments of flYB5 and 212bp 307 bp fMYBd9 (2,439 and 2,405 bp, respectively) due to the 34-bp deletion is notresolved by this gel. The f1YE3 (lane E) andflYEdl (lane G)HgiAI fragment (379 and 344 bp, respectively) mobilities indicated that their orientations are identical. The difference in mobilities is due to the 35- Insertion back into flR229 bp deletion in flYEd1 DNA. 2384__2350 2121 2154 recovered by electrophoresis on acrylamide gels followed by EcoRI EcoRI EcoRIlt_-VEcoRI electroelution. The purified DNA fragments were then di- gested with the appropriate enzyme (Hha I or Msp I), labeled with 32P by using either T4 polynucleotide kinase or DNA poly- flYEdl flYBd9 merase I, and displayed on 10% polyacrylamide gels (Fig. 3). The absence ofa 34-base pair (bp) DNA fragment in lane D that was present in lane B confirmed the predicted size of 34 nucleo- FIG. 1. Construction of factor Y effector site deletions. RFI tides for the deletion in f1YBd9 DNA. However, unlike the [3H]DNAs of recombinant phages f1YE3 and flYB5 were cleaved with f1YE3 DNA fragment, the fMYEdl EcoRI DNA fragment could EcoRI, treated with bacterial alkaline phosphatase, and electropho- no longer be-cleaved by Hha I (lane H). Analysis ofthe total Hha resed through a 5% polyacrylamide gel (30:1 acrylamide/bisacryla- I restriction pattern off1YE3 RFI DNA and the series ofdeleted mide). The inserted DNA fragments were recovered by-electroelution. f1YE3 RFI DNAs confirmed this result (data not shown). The f1YE3 and f1YB5 insert DNA fragments were cleaved by Hha I The reason for this anomaly was revealed when the and Msp I, respectively, and passed a nucleo- separately through Sephadex G- tide sequence in the deleted regions was 50 column (0.8 x 95 cm) developed in 100 mM NaCl/20 mM Tris HCl, determined (data not pH 8 (at 4°0)/1 mM EDTA to remove the smaller, central restriction shown). The sequence data for flYBd9 confirmed that a 34-bp fragments. Excluded fractions were detected by scintillation counting, DNA fragment extending from pBR322 nucleotide residues pooled, concentrated by sec-butyl alcohol extraction, and ethanol pre- 2,121 to 2,154 had been removed from the H strand factor Y cipitated. The DNA fragments were resuspended in 2 A.l of ligation site (Fig. 4). As expected, ligation ofthe flanking areas resulted buffer, 1.0 unit of T4 polynucleotide ligase was added, and the mixture in the regeneration of a single Msp I site (Fig. 3). Comparison was incubated at 400 for 24 hr. The ligated fragments were labeled at the 5' ends with 32P by using [y-32P]ATP andT4 polynucleotide kinase ofthe flYEdl DNA sequence with that ofpBR322 showed the and joined to EcoRI-linearized flR229 RF vector DNA. The resultant presence of a 35-bp deletion extending from pBR322 nucleo- mixture was transfected into E. coli K38 and plated on lawns of K38 tide residues 2,350 to 2,384 in flYEdl DNA (Fig. 4). Appar- indicator bacteria. Recombinant bacteriophage were detected by ently, a contaminating exonuclease activity removed the Hha plaque hybridization (30). Recombinant molecules having the correct I-generated sticky ends, resulting in a deletion 2 bp longer than orientation ofthe inserted fragment were identified as described in the legend to Fig. 2. The scale at the top refers to nucleotide positions in expected. Thus, no Hha I site was regenerated when the flank- pBR322 DNA (32): dashed line, pBR322 H strand; heavy solid line, ing ends were joined. pBR322 L strand; light solid line, fl (+) strand. Restriction sites on Factor Y ATPase Effector Activity of the Deleted DNAs. the recombinant molecules are indicated by: -*, BamHI; -4, HgiAI; E. coli replication factor Y has been shown to possess a se- and -, Pvu II. quence-specific ss DNA-dependent ATPase activity (15, 19). Downloaded by guest on September 26, 2021 7256 Biochemistry: Soeller and Marians Proc. Nad Acad. Sci. USA 79 (1982) Table 1. ATPase effector activity of recombinant fl phage ss(c) A B C D E F G H I DNAs and their deletion derivatives DNA 32P, formed, added nmol/10 min

.. la 4X174 3.20 f1R229 0.05 flYB5 1.77 a 0 flYBM 0.03 fMYBd9 0.01 f1YE3 1.63 f1YE5 0.05 flYEdl 0.01

mosensitive strains requires the addition of the respective pu- rified wild-type proteins. The recombinant phage DNAs flYB5 A.~U and f1YE3 have been shown to serve as efficient templates for rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent DNA synthesis (16). We compared their template activity to that of their respective deletion mutants. The addition ofthe purified dnaB, dnaC, or dnaG gene prod- FIG. 3. Restriction analysis of EcoRI inserts. Lanes: A, the f1YB5 ucts to reaction mixtures containing their respective ammonium EcoRI insert; B, flYB5 EcoRI insert cleaved with Msp I; C, f1YBd9 sulfate receptor fractions and either 4X174, fMYE3, or flYBM EcoRI insert; D, flYBd9 EcoRI insert cleaved with Msp I; E, f1YE3 ss(c) DNA as template resulted in a 25- to 100-fold stimulation EcoRI insert; F, f1YE3 EcoRI insert cleaved with Hha I; G, flYEd& EcoRI insert; H, flYEdl EcoRI insert cleaved with Hha I; I, f1YE3 of the rate of DNA synthesis (Table 2). In contrast, DNA syn- HaeJl fragments as markers. EcoRI insert fragments and their Msp thesis was stimulated 0- to 10-fold by the addition of the ap- I digestion products were labeled by repair synthesis using [a- propriate purified gene product when flR229, flYBi, or f1YE5 32PldATP, [a-32P]dCTP, and DNApolymerase I.HaeHIandHha Ifrag- ss(c) DNA was used as template. This residual stimulation may ments were treated with bacterial alkaline phosphatase and then la- be due to a combination ofgeneral priming characteristic ofthe beled at the 5' ends by using [y-32P]ATP andT4 polynucleotide kinase. dnaB and dnaG gene products and to endonuclease activity Labeled fragments were electrophoresed through a 10% polyacryla- mide gel (30:1 acrylamide/bisacrylamide) in TBE buffer at 15 V/cm present in the receptor fraction which may nick the template for 60 min. An autoradiograph of the dried gel is shown. DNA, allowing elongation from foldback structures. The stim- ulation of DNA synthesis observed when the deletion mutants We tested various ss(c) DNAs for their ability to act as effectors were used as templates closely resembled that observed when for this activity (Table 1). flYBl and fLYE5 are recombinant fl the vector DNA or the inactive parental phage DNA was used. DNAs that lack effector activity. These phages contain the com- These data indicate that some portion of the sequences deleted plement ofthe pBR322 sequences carried by f1YB5 and fLYE3 are required for the factor Y sites to function as rifampicin-re- DNAs (16). flYB5 and f1YE3 ss(c) DNA supported 55% and sistant dnaB-, dnaC-, and dnaG-dependent origins of replica- 51%, respectively, ofthe ATPase activity observed with 4X174 tion. ss(c) DNA as the effector. In contrast, flYBd9 and flYEdl ss(c) DNAs were inactive as effectors for the factor Y ATPase activity. DISCUSSION Mixing experiments confirmed that the lack ofeffector activity We previously reported (13) that pBR322 DNA contained two was not due to any inhibitory factor present in the ss(c) DNA factorY effector sites, located on opposite strands near its origin preparations (data not shown). Therefore, we conclude that at of replication. Cloning of small pBR322 DNA fragments con- least some ofthe sequences present within nucleotide residues taining these sites into flR229 DNA allowed us to demonstrate 2,121-2,154 and 2,350-2,384 in pBR322 DNA are essential for that these regions could function as origins ofreplication in ss(c) factor Y ATPase effector activity. DNA -- RF DNA synthesis via a 4X174-like rifampicin-resist- DNA Synthesis in Receptor Fractions Prepared from E. coli ant dnaB-, dnaC-, and dnaG-dependent pathway (16). These Thermosensitive DNA Replication Mutants. DNA synthesis results are consistent with those ofNomura and Ray who dem- using 4X174 and fl recombinant phage ss(c) DNAs as templates onstrated that M13 ss(c) DNA containing the L strand ofColEl was examined with ammonium sulfate receptor fractions pre- Hae II fragment E could be converted to the duplex replicative pared from E. coli strains thermosensitive in the dnaB form via a rifampicin-resistant dnaB- and dnaG-dependent (BT1029), dnaC (PC22), and dnaG (NY73) genes. ss(c) DNA mechanism in vivo (33). In addition, Boldicke et aL were able -- RF DNA synthesis using receptor fractions from such ther- to convert the isolated H and L strands of ColEl DNA to the

pBR322 H strand site 5'-CAG CT CC|CGGAGACGGTCACAG CTTGTCTGTAAGCGGATGCICGGGAG CAGACAAG CCCGT CAGGG CGCGT CA -3'

pBR322 L strand site 5'-CTGATACCGCTCGCCGCAGCCGAACGAC'CGAGjCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGC -3'

FIG. 4. Sequence of the deleted Y sites (shown in boxes). The nucleotide coordinates (32) of the sites as shown are 2,114-2,185 for the H strand and 2,416-2,349 for the L strand. The maximal limits of the H strand Y site correspond to the sequence shown; the limits for the L strand are 2,416- 2,353 (14). Downloaded by guest on September 26, 2021 Biochemistry: Soeller and Marians Proc. Natl. Acad. Sci. USA 79 (1982) 7257

Table 2. Requirement for dnaB, dnaC, and dnaG in ss(c) -+ RF We thank Dr. J. Hurwitz and Dr. J. Marmur for a critical reading DNA synthesis of 4X174 and various recombinant fl DNAs of the manuscript and J. Greenbaum for teaching one of us (W.C.S.) TMP incorporated, pmol/15 min DNA sequence analysis. This work was supported by Grant GM26410 from the National Institutes of Health and Grant JFRA-14 from the BT1029 PC22 NY73 American Cancer Society to K.J.M. and by Grant INT 8018468 from Template dnaB dnaC dnaG the National Science Foundation to K.J.M. and J. Hurwitz. W.C. S. was supported by National Institutes of Health Training Grant T32- DNA Without With Without With Without With CA09060-07. 4X174 0.6 58.1 2.8 61.2 1.2 41.8 f1R229 1.6 1.5 0.8 2.7 2.1 1.8 1. Wickner, W., Brutlag, D., Schekman, R. & Kornberg, A. (1972) flYB5 0.9 24.6 0.6 47.9 Proc. Natl Acad. Sci. USA 69, 965-969. 1.7 45.0 2. Vicuna, R., Hurwitz, J., Wallace, S. & Girard, M. (1977)J. Biol flYBi 0.3 3.7 0.7 5.1 1.5 2.3 Chem. 252, 2524-2533. flYBd9 0.9 2.3 1.1 4.1 1.9 3.4 3. Vicuna, R., Ikeda, J.-E. & Hurwitz, J. (1977)J. Biol Chem. 252, f1YE3 1.4 52.5 0.8 58.7 1.8 59.3 2534-2544. f1YE5 1.1 1.8 0.6 5.1 1.0 3.1 4. Kaguni, J. M. & Kornberg, A. (1982) 1. Biol Chem. 257, 5437- flYEdl 0.7 3.7 0.9 5.2 1.6 3.3 5443. 5. Bouche, J.-P., Zechel, K. & Kornberg, A. (1975) J. Biol Chem. Optimal concentrations of receptor fractions and purified gene prod- 250, 5995-6001. ucts were determined for each template. Template DNAs were at equi- 6. Bouche, J.-P., Rowen, L. & Kornberg, A. (1978) J. Biol Chem. molar concentrations (2.2 pmol/ml). 253, 765-769. 7. Sims, J. & Benz, E. W., Jr. (1980) Proc. Natl Acad. Sci. USA 77, duplex form by a rifampicin-resistant pathway involving the 900-904. dnaB, dnaC, and dnaG gene products in vitro (34). 8. Arai, K. & Kornberg, A. (1981) Proc. Natl Acad. Sci. USA 78, 69- Factor Y dictates the assembly ofthe primosome at a specific 73. 9. Wickner, S. & Hurwitz, J. (1974) Proc. Natl Acad. Sci. USA 71, site on the DNA. Thus, in order to understand the signals en- 4120-4124. coded in the DNA that are necessary for the proper initiation 10. Schekman, R., Weiner, J. H., Weiner, A. & Kornberg, A. (1975) of DNA replication we have undertaken a detailed analysis of J. Biol Chern. 250, 5859-5865. the interaction between factor Y and its effector sites. We report 11. Low, R. L., Arai, K. & Kornberg, A. (1981) Proc. Nati Acad. Sci. here the construction and characterization of deletion mutants USA 78, 1436-1440. in the factor Y effector sites of pBR322 DNA. These deletions 12. Arai, K., Low, R. L. & Kornberg, A. (1981) Proc. Nati Acad. Sci. extend from USA 78, 707-711. pBR322 nucleotide residues 2,121-2,154 in the H 13. Zipursky, S. L. & Marians, K. J. (1980) Proc. Natl Acad. Sci. USA strand and residues 2,350-2,384 in the L strand. Both deletions 77, 6521-6525. fall within regions previously shown to be active as ATPase ef- 14. Marians, K. J., Soeller, W. & Zipursky, S. L. (1982) J. Biol. fector DNA sequences for factor Y. Chem. 257, 5656-5662. We have demonstrated that, in contrast to the parental sites 15. Shlomai, J. & Kornberg, A. (1980) Proc. Nati Acad. Sci. USA 77, from which they were derived, both deletion mutants were in- 799-803. active 16. Zipursky, S. L. & Marians, K. J. (1981) Proc. Natl Acad. Sci. USA both as effectors for E. coli factor Y ATPase activity and 78, 6111-6115. as rifampicin-resistant dnaB-, dnaC-, and dnaG-dependent 17. Kabori, J. & Kornberg, A. (1982) J. Biol Chem., in press. origins ofreplication. Thus, the DNA regions previously char- 18. Boeke, J. E. (1981) Mol Gen. Genet. 181, 288-291. acterized functionally as factor Y sites have now been so defined 19. Wickner, S. & Hurwitz, J. (1975) Proc. Nati Acad. Sci. USA 72, by mutational analysis. The sequences ofthe regions containing 3342-3346. the Y sites of pBR322 are shown in Fig. 4. Both deletions re- 20. Shlomai, J. & Kornberg, A. (1980)J. Biol Chem. 255, 6789-6793. 21. Reha-Krantz, L. J. & Hurwitz, J. (1978)J. Biol Chem. 253, 4043- move a hexanucleotide sequence 5' A-A-G-C-G-G 3' previous- 4049. ly shown to be common to the pBR322 and 4X174 Y sites (14) 22. Lanka, E., Edelbluth, C., Schlicht, M. & Schuster, H. (1978)J. which may be involved in factor Y recognition. The formal pos- Biol, Chem. 253, 5847-5851. sibility exists that the sequences deleted in flYBd9 and flYEdl 23. Benz, E. W., Jr., Reinberg, D., Vicuna, R. & Hurwitz, J. (1980) serve as spacer sequences which separate flanking recognition J. Biol Chem. 255, 1096-1106. sites by a critical distance. However, this is unlikely because 24. Francke, B. & Ray, D. (1970) Virology 44, 168-187. we have determined that factor Y protects five bases from nu- 25. Zinder, N. D. & Boeke, J. E. (1982) Gene 19, 1-10. clease 26. Model, P. & Zinder, N. D. (1974)J. Mol Biol 83, 237-251. cleavage within the deleted sequence ofthe H strand site 27. Conway, T. & Lipmann, F. (1964) Proc. Natl Acad. Sci. USA 52, (unpublished data). Thus, the 34 nucleotides deleted from the 1462-1469. H strand Y site may be the region of primary interaction be- 28. Mandel, M. & Higa, A. (1970)J. Mol Biol 53, 159-162. tween factor Y and the DNA. The coinactivation ofboth Y site- 29. Benton, W. D. & Davis, R. W. (1977) Science 196, 180-182. catalyzed activities described here-ATPase effector activity 30. Wahl, J. M., Stem, M. & Stark, G. R. (1979) Proc. Nati Acad. and origin function-by the same mutational event reveals the Sci. USA 76, 3683-3687. intimate association between the site-specific DNA-dependent 31. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) ATPase activity of factor Y and the initiation of lagging-strand 1. MoL BioL 113, 237-251. DNA replication. 32. Sutcliffe, J. G. (1978) Cold Spring Harbor Symp. Quant. Biol. 43, The role of 77-90. the factor Y sites in pBR322 DNA replication and 33. Nomura, N. & Ray, D. S. (1980) Proc. Natl Acad. Sci. USA 77, the functional configuration of these sites are not yet known. 6566-6570. Isolation of smaller deletions and base substitution mutants 34. Boldicke, T. W., Hellinbrand, G., Lanke, E. & Staudenbauer, within these sites should allow us to answer these questions. W. L. (1981) Nucleic Acids Res. 9, 5215-5231. Downloaded by guest on September 26, 2021