Rnase H Confers Specificity in the Dnaa-Dependent Initiation

Home , DnaA, DNA gyrase, Suppressor mutation

Proc. Nat!. Acad. Sci. USA Vol. 81, pp. 1040-1044, February 1984 Biochemistry

RNase H confers specificity in the dnaA-dependent initiation of replication at the unique origin of the Escherichia coli chromosome in vivo and in vitro (rnh/sdrA/dasF/oriC) TOHRU OGAWA*, GAVIN G. PICKETTt, TOKIo KOGOMAt, AND ARTHUR KORNBERG* *Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305; and tDepartment of Biology, The University of New Mexico, Albuquerque, NM 87131 Contributed by Arthur Kornberg, October 31, 1983

ABSTRACT Escherichia coli rnh mutants defective in (dasF) of dnaAts mutations has been located near proA (21). RNase H activity display the features of previously described The dasF mutants exhibit the Sdrc phenotype (unpublished sdrA (stable DNA replication) and dasF (dnaA suppressor) mu- observations). These several results suggest the presence of tants: (i) sustained DNA replication in the absence of protein an alternative initiation pathway, distinct from the dnaA+- synthesis, (ii) lack of requirement for dnaA protein and the and oriC+-dependent system, and a suppression of this alter- origin of replication (oriC), and (iii) sensitivity of growth to a native pathway under normal growth conditions by the rich medium. Both the sdrA mutants (selected for continued sdrA+ (dasF+) gene product. DNA replication in the absence of protein synthesis) and the In this report, we present evidence that both the sdrA and dasF mutants (selected as dnaA suppressors) are defective in dasF genes are identical with the rnh gene, which encodes RNase H activity, measured in vitro. Furthermore, a 760-base- RNase H, and that RNase H is a specificity protein in pair fragment containing the rnh+ structural gene comple- dnaA+- and oriC+-dependent DNA replication in E. coli. ments the phenotype of each of the rnh, sdrA, and dasF mutants, indicative of a single gene. One function of RNase H in MATERIALS AND METHODS vivo is in the initiation of a cycle of DNA replication at oriC The E. coli strains used are listed in Table 1. The levels of dependent on dnaA+. In keeping with these results, RNase H RNase H in extracts of ON121(rnh-59) are 0.1% and in contributes to the specificity of dnaA protein-dependent repli- ON152(rnh-91) less than 0.05% of the wild-type level (22). cation initiated at oriC in a partially purified enzyme system. Plasmid pSK760 (24), a derivative of pBR322 that carries a 760-base-pair sequence of E. coli DNA including the RNase Replication of the Escherichia coli chromosome starts at a H gene, was obtained from R. Crouch (National Institutes of unique site (oriC) and proceeds bidirectionally (1-3). The Health). DNA fragment containing oriC has been cloned and se- Media used were M9 salts/glucose (25) and L broth (LB quenced (4, 5). Subsequent analyses of the structure and in medium) (26). Depending on the strains used, required sup- vivo function of the oriC region have disclosed that a 245- plements, in ,g/ml, included: thymine, 8; histidine, 20; tryp- base-pair sequence is required and that within this region tophan, 20; arginine, 100; asparagine, 20; thiamin-HCI, 3; certain unique sequences surrounded by defined stretches of and for the plasmid-containing strains, ampicillin, 50. spacer sequences must be strictly maintained (6, 7). RNase H activity in extracts was assayed with [3H]poly(C)Q The initiation of replication at oriC is dependent on several poly(dG) as substrate (22, 27). Extracts were prepared by gene products, particularly dnaA (for review, see ref. 8). The lysing the cells (grown to late logarithmic or stationary phase dnaA protein, purified to near homogeneity (9), is an essen- and washed once with 50 mM Tris'HCl, pH 8.0/10% su- tial component required at an early stage of the initiation re- crose) in a solution containing 50 mM Tris HCl at pH 8.0, action in an in vitro system which replicates oriC plasmids 10% sucrose, 300 mM KCl, 2 mM dithiothreitol, 2 mM (10, 11) and binds a 9-base-pair sequence which appears four EDTA, egg lysozyme at 250 ug/ml, phage T4 lysozyme at times in oriC. The dnaB and dnaC gene products, essential 0.1 ,g/ml, and 6 x 109 to 6 x 1010 cells per ml for 30 min at for ongoing replication, are also required during or shortly 0°C followed by 2 min at 30°C. The debris was removed by after initiation (12, 13). RNA polymerase has also been im- centrifugation at 10,000 x g for 10 min. One unit of RNase H plicated by the inhibitory effect of rifampicin at this stage of activity was defined as the amount that released 1 nmol of replication (14) and by a dnaA suppressor identified with trichloroacetic acid-soluble radioactive material in 15 min. rpoB, the l3-subunit gene of RNA polymerase (15). Protein concentration was determined (28) with bovine se- The initiation of each new round of replication of the E. rum albumin as a standard. Transformation with plasmid coli chromosome requires de novo protein synthesis (14, 16, DNA was carried out as described (26). 17). The biochemical nature of this requirement for protein In vitro reactions (25 ,ul) for DNA replication were for 30 synthesis is still unknown. Mutants, termed constitutive sta- min at 30°C as described (29, 30). Proteins included were: ble DNA replication (Sdrc) mutants, have been isolated that protein HU, topoisomerase I, DNA gyrase, RNA polymer- maintain DNA synthesis despite the absence of protein syn- ase, single-stranded DNA-binding protein, dnaB and dnaC thesis (stable DNA replication) (18, 19). One of these mutant proteins, primase, DNA polymerase III holoenzyme, and alleles (sdrA224) maps at 5 min between metD and proA (un- flow-through and 0.5 M KCl eluate fractions obtained by published observations). The sdrA224 mutant tolerates trans- Amicon red-A agarose column chromatography of a crude poson insertional inactivation of the dnaA gene or oriC de- enzyme fraction. dnaA protein and RNase H were added as letion (20). A group of extragenic suppressor mutations specified.

The publication costs of this article were defrayed in part by page charge RESULTS payment. This article must therefore be hereby marked "advertisement" The rnh Mutants Exhibit the Sdrc Phenotype. Observations in accordance with 18 U.S.C. §1734 solely to indicate this fact. that RNase H contributes to the specificity of a dnaA and

1040 Downloaded by guest on September 27, 2021 Biochemistry: Ogawa et aL Proc. NatL. Acad. Sci. USA 81 (1984) 1041 Table 1. E. coli K-12 strains press the stable DNA replication of the rnh-91 mutant. These Strain* Relevant gen4Lotype Source/ref. results indicate that the observed Sdrc phenotype is caused by rnh mutations. Some increase of the DNA content in plas- ON112 (a) rnh+ proA (22) mid-containing strains may be attributable to replication of ON121 (a) rnh-59 (22) the plasmids that occurs even in the presence of chloram- ON152 (a) rnh-91 (22) phenicol (33). AQ634 (b) sdrA+ (23) The rnh Mutant Shares a Common Phenotype with sdrA and AQ666 (b) sdrA224 pro' (23) dasF Mutants. Since the sdrA and dasF mutations map near AQ685 (c) sdrA' (20) rnh (5 min on the E. coli chromosome) (18, 21) and all these AQ699 (c) sdrA224 proA+ (20) mutations cause the Sdrc phenotype, we examined the possi- AQ1355 (d) dasF373 dnaA5 proA+ P1TC373 (21) x DK175, ble identity of these three genes. In addition to constitutive select pro stable DNA replication (Sdrc), sdrA and dasF mutants ex- AQ1363 (d) dasF377 dnaAS proA+ P1TC373 (21) x DK175, hibit three other phenotypic features in common: Das (sup- select pro+ pression of dnaA), Dos (suppression of AoriC), and Srm AQ1722 (c) sdrA225 proA+ dnaA5 Transformant of DK83 (sensitivity to rich media) (ref. 20; unpublished observa- tnaA600: :TnlO tions). The rnh mutants were examined for these additional (pSK760) phenotypic features. As shown in Fig. 2, the rnh-91 mutation AQ1724 (d) dasF373 dnaA5 metD+ Transformant of DK282 suppressed the temperature sensitivity of dnaA5 when trans- (pSK760) ferred into DK175. This Das activity was 13% linked to AQ1732 (d) rnh-91 dnaA5 metD+ P1ON152 x DK175, se- metD. The rnh-91 mutation also suppressed dnaA850::TnJO lect metD+ transferred into ON152. The Das phenotype of rnh-91 mu- AQ1760 (d) rnh-91 dnaA5 metD+ Transductant of AQ1732 tant (AQ1732) was complemented by pSK760 (Fig. 2). The AoriC1071 with Xasn132 rnh-91 mutation also enabled cells to dispense with the oriC AoriCIO71 (20) sequence, although growth was significantly perturbed. AQ1773 (d) rnh-91 dnaA5 metD+ Transformant of AQ1732 AQ1760 in Fig. 2 is an example of AoriC rnh-91 constructed (pSK760) by the procedure reported previously (20). AQ1789 (c) sdrA225 proA+ dnaA5 Transformant of DK83 ON152 (rnh-91) is not sensitive to rich media (22). Howev- tnaA600: :TnlO er, when the rnh mutation was transferred into DK175 by (pBR322) selection for the Das phenotype, all the Das- clones were AQ1790 (d) dasF373 dnaA5 metD+ Transformant of DK282 sensitive to rich media (Srm-). It appears that ON152 con- (pBR322) AQ1791 (d) rnh-91 dnaA5 metD+ Transformant of AQ1732 (pBR322) DK83 (c) sdrA224 proA + dnaA5 P1TC743 (20) x AQ699, tnaA600: :TnlO select tetracycline resistance DK175 (d) sdrA+ dnaA5 (20) DK241 (c) sdrA224 proA+ asnB50: :Tn5 (20) AoriClO71 DK249 (c) sdrA224 proA + (20) dnaA850::TnlO DK282 (d) dasF373 dnaA5 metD+ P1Tc373 (21) x DK175, z select metD+ 0 thi endA C]0 *Remaining genotypes are as follows: (a) F- metE val rna 4- supE; (b) F- argH ilv metB his-29 trpA9605 pro thyA deoB (or C); c (c) F- argH metBl his-29 trpA9605 proA3 metD88 thyA deoB (or E C) rpoB; (d) F- argH metBI his-29 trpA9605 proA3 metD88 thyA Ca deoB (or C) rpoB ilv-192 asnB50::Tn5. a)

oriC-dependent DNA replication system in vitro (see Table 3 -cr below; also, unpublished observations) and the ability of an sdrA mutant to dispense with dnaA gene function and the oriC sequence (20) prompted us to test rnh mutants for the Sdrc phenotype. The amount of DNA in cultures of two rnh mutants [ON121 (rnh-59) and ON152 (rnh-91)] and of the wild-type strain (ON112) was measured after inhibition of protein synthesis (Fig. 1). In the wild-type strain, DNA synthesis stopped 2 hr after inhibition of protein synthesis. During this period, the DNA content increased by about 40%, as expect- ed from continued replication until the terminus was reached. In contrast, the rnh- mutants continued synthesis for at least 7 hr after inhibition of protein synthesis, and the Time in chloramphenicol, hr of DNA content increased by 150% or more. Introduction FIG. 1. Stable DNA replication in rnh mutants. Cells were plasmid pSK760, which contains the rnh gene, suppressed grown at 37°C in M9/glucose medium. At about 2 x 108 cells per ml, the stable DNA replication in the rnh mutants. This plasmid, chloramphenicol was added at 10 ,ug/ml. At indicated times, 1-ml a derivative of pBR322, contains a 760-base-pair fragment of portions of the culture were withdrawn and the amount of DNA was the E. coli chromosome covering the entire rnh gene and part estimated (31). (A) ON112 (wild type), (B) ON121 (rnh-59), and (C) of the dnaQ gene (24, 32). The vector, pBR322, did not sup- ON152 (rnh-91). Downloaded by guest on September 27, 2021 1042 Biochemistry: Ogawa et aL Proc. NatL. Acad Sci. USA 81 (1984) 30°C 42 C

M9 Glucose

L Broth

FIG. 2. Presence or absence of colony formation of rnh, sdrA, and dasF mutants on minimal or broth plates at 30'C or 420C. Mutant cells were allowed to form colonies on M9/glucose plates at 30'C. Cells of a single colony from these plates were inoculated onto two M9/glucose and two L broth plates and spread with toothpicks. One M9/glucose and one L broth plate were incubated at 30'C (A, C) and the others at 420C (B, D). The M9/glucose plates (A, B) and L broth plates (C, D) were incubated for 48 and 24 hr, respectively. Spread strains: 1, AQ685 (wild type); 2, DK175 (dnaA5); 3, DK83 (sdrA224 dnaA5); 4, DK282 (dasF373 dnaA5); AQ1732 (rnh-91 dnaA5); 6, AQ1722 [sdrA224 dnaA5 (pSK760)]; 7, AQ1724 [dasF373 dnaA5 (pSK760)]; 8, AQ1773 [rnh-91 dnaA5 (pSK760)]; 9, AQ1789 [sdrA224 dnaA5 (pBR322)]; 10, AQ1790 [dasF373 dnaA5 (pBR322)]; 11, AQ1791 [rnh-91 dnaA5 (pBR322)]; and 12, AQ1760 (rnh-91 dnaA5 AoriC1071).

tains a suppressor mutation (sis) (unpublished observations) dasF373 mutants (Fig. 3). The vector, pBR322, did not affect that specifically alleviates the Srm- phenotype of sdrA mu- the Sdrc phenotype of the sdrA224 mutant (data not shown). tants. The Srm- phenotype of AQ1732 (rnh-91) was comple- The Srm- phenotype of sdrA and dasF mutants was also mented by pSK760 (Fig. 2). suppressed by pSK760 (Fig. 2). The sdrA and dasF Mutants are Defective in RNase H Activ- Complementation of the Das phenotype of sdrA and dasF ity. Inasmuch as the rnh mutants possess the phenotype of showed certain complexities (Fig. 2): on rich plates, the sdrA sdrA and dasF mutants, we examined the RNase H activity dnaA and dasF dnaA double mutants were completely tem- in extracts of sdrA and dasF mutants. As shown in Table 2, perature sensitive, although they grew well at 30°C (i.e., RNase H activity was undetectable in all sdrA and dasF mu- Srm+); however, they were not clearly temperature sensi- tants tested. Introduction of pSK760 into two of these mu- tive on minimal plates. Measurement of [3H]thymine incor- tants, as well as into the wild-type strain, resulted in a 10- poration into DNA revealed that, although the rate of DNA fold enhancement over the wild-type level. synthesis in these mutants carrying pSK760 is much slower The rnh+ Plasmid Complements all the Phenotypic Features at 42°C than that of the strains not carrying the plasmid, of sdrA and dasF Mutants. The rnh+-containing plasmid, DNA synthesis continued at a slower rate for a considerable pSK760, corrected the Sdrc phenotype of sdrA224 and period of time (data not shown). Thus, the extent of comple- Downloaded by guest on September 27, 2021 Biochemistry: Ogawa et aL Proc. Natl. Acad. Sci. USA 81 (1984) 1043

Table 2. RNase H activity of sdrA and dasF mutants Table 3. Specificity activity of RNase H in oriC replication Activity, in vitro Strain Plasmid units/mg DNA synthesis, pmol AQ634 (wild type) None 32 dnaA protein RNase H M13oriC26 RF I OX174 RF I pSK760 386 - - 214 434 AQ666 (sdrA224) None <0.05 - + 13 14 pSK760 351 + - 468 348 DK241 (sdrA224 AoriCJ071) None <0.05 + + 351 34 DK249 (sdrA224 dnaA850::TnJO) None <0.05 AQ1355 (dnaAS dasF373) None <0.05 Reactions using replicative form (RF) I DNA of M13oriC26 (a chi- pSK760 327 meric M13 phage containing the E. coli oriC+ sequence) (34) or of AQ1363 (dnaA5 dasF377) None phage 4X174 as template (600 pmol) were in the presence (+) or <0.05 absence (-) of dnaA protein (50 ng) and RNase H (2 ng). DISCUSSION mentation of the Das phenotype by pSK760 appears to be allele specific under certain conditions. In this report, we have shown that rnh (RNase H) mutants RNase H is a Specificity Factor for Replication of oriC DNA exhibit several distinctive phenotypic features: Sdrc (consti- in Vitro. Ability of rnh (sdrA, dasF) mutants to dispense with tutive stable DNA replication), Srm (sensitivity to rich me- dnaA+ and oriC+ functions suggests the presence of a sec- dia), Das (dnaA- suppression), and Dos (oriC- suppres- ond pathway of initiation of replication alternative to the sion). sdrA mutants, isolated as mutants capable of constitu- dnaA+- and oriC+-dependent mechanism. It appears that, in tive stable DNA replication, and dasF mutants selected for wild-type cells, RNase H prevents the use of this alternative their ability to suppress dnaAt's mutations, were defective in pathway. This role of RNase H was directly demonstrated in RNase H activity (Table 2). Furthermore, the phenotypes vitro (Table 3). In the absence of RNase H, efficient replica- due to each of these three independent mutations were com- tion took place on supercoiled replicative form (RF) I DNA plemented by a 760-base-pair segment of E. coli chromo- of phage 4X174 as well as on that of phage M13 oriC26 (con- some containing the rnh gene carried by pSK760. This se- taining the E. coli oriC+ sequence) both in the presence and quenced segment (24) contains no complete gene except that absence of the dnaA protein. Addition of RNase H severely for RNase H. Thus the sdrA, dasF, and rnh genes appear to inhibited 4X174 DNA synthesis but not that of the dnaA+- be identical. and oriC+-dependent reaction. These findings have indicated a role for RNase H in the initiation of replication of the E. coli chromosome. The fact that rnh mutants can survive the absence of the dnaA function and the oriC sequence implies that chromosomal repli- A. WILD TYPE PLASMID cation can be initiated at other sites in a dnaA-independent pSK760 mechanism and that, in wild-type cells, RNase H prevents 2 _ _ the use of this alternative initiation system. This specificity ~~~~~~~~~None activity of RNase H was directly demonstrated in vitro (Ta- x ble 3). Discriminatory actions by RNase H have also been E reported in vitro in replicon-specific replication of fd viral o DNA (35) and ColEl DNA (36, 37). The simplest explana- .o 4 _ B. sdrA224 tion for the role of RNase H is that it eliminates an RNA transcript hybridized to template DNA that would allow ab- 0 normal priming of DNA replication. This explanation does 0. o not exclude a more direct involvement of RNase H in dnaA- .' 2 pSK760_ and oriC-dependent initiation, for example, in the creation of a correct primer terminus for DNA synthesis as in the ColEl system (38). Another possible role for RNase H is in the re- moval of the 5' terminus of longer primer RNA linked to the 0 nascent short DNA (Okazaki) fragments during discontinu- ous DNA replication (22). c/ 4- C. dasF373 dnaA5 None At present, little is known about the molecular mechanism of initiation of replication in rnh (sdrA) mutants. DNA replication in these mutants is recA+ independent unless protein C') synthesis is inhibited (39), suggesting the presence of a < 2-__ dnaA-dependent oriC-dependent replication system (see below). Operation of the stable replication pathway would also 0/ pSK760 be possible under recA+ conditions. However, the broth sensitivity of these mutants suggests that replication is not as 0 efficient as in wild-type cells. Under dnaA- oriC- condi- 0 2 4 6 tions, the rnh mutants depend totally on the stable replica- Time in chloramphenicol, hr tion pathway. Slow growth of the rnh AoriC mutant suggests the inefficiency of this pathway. FIG. 3. Complementation of Sdrc phenotype of sdrA224 and Genetic studies suggest that stable replication is one of the dasF373 mutants by pSK760. Cells were grown in M9/glucose medi- recA+ lexA+-dependent SOS functions (40, 41). In wild-type um at 37°C (A, B) or 320C (C) to about 1.5 x 108 cells per ml. Chlor- stable treatments amphenicol and [3H]thymine were added at final concentrations of cells, replication can be induced by several 250 ,ug/ml and 4 ,Ci/ml, respectively (1 Ci = 37 GBq). At indicated that induce SOS functions. Protease-related activity of recA times, a 0.5-ml portion of the culture was withdrawn and radioactiv- protein may be required to establish stable replication, and ity in the acid-insoluble fraction was measured. (A) AQ634 (wild maintenance of stable replication may depend on the recom- type); (B) AQ666 (sdrA224); (C) AQ1355 (dasF373 dnaAS). bination activity of recA protein (39, 40). A better under- Downloaded by guest on September 27, 2021 1044 Biochemistry: Ogawa et aL Proc. Nati. Acad. Sci. USA 81 (1984)

standing is needed of the events leading to stable replication Biology, eds. Ray, D. S. & Fox, C. F. (Academic, New York), in wild-type cells and of the relationship of induced stable Vol. 21, pp. 881-889. in cells to constitutive stable 20. Kogoma, T. & von Meyenburg, K. (1983) EMBO J. 2, 463- replication wild-type replica- 468. tion in rnh (sdrA) mutants. 21. Atlung, T. (1981) in The Initiation ofDNA Replication, ICN- UCLA Symposia on Molecular and Cellular Biology, eds. Ray, We thank Robert Crouch for plasmid pSK760, Tove Atlung for D. S. & Fox, C. F. (Academic, New York), Vol. 21, pp. 297- strains and Ted Torrey for excellent technical assistance. We also 314. thank Jon Kaguni, Nicholas Dixon, Robert Fuller, and LeRoy 22. Ogawa, T. & Okazaki, T. (1983) Mol. Gen. Genet., in press. Bertsch for the oriC reconstitution assay. This work was supported 23. Kogoma, T. & Subia, N. L. (1983) in Mechanism ofDNA Rep- by grants from the National Institutes of Health to A.K. (GM 07581) lication and Recombination: UCLA Symposia on Molecular and T.K. (GM 22092 and Minority Biomedical Research Support and Cellular Biology, New Series, ed. N. R. Cozzarelli (Liss, Grant PR08139) and from the National Science Foundation to A.K. New York), Vol. 10, in press. (PCM 82-09380A1). 24. Kanaya, S. & Crouch, R. J. (1983) J. Biol. Chem. 258, 1276- 1281. 1. Bird, R. E., Louarn, J., Martuscelli, J. & Caro, L. (1972) J. 25. Lark, K. G., Repko, T. & Hoffman, E. G. (1963) Biochim. Mol. Biol. 70, 549-566. Biophys. Acta 76, 9-24. 2. Masters, M. & Broda, P. (1971) Nature (London) New Biol. 26. Davis, R. W., Botstein, D. & Roth, J. R. (1980) Advanced 232, 137-140. Bacterial Genetics (Cold Spring Harbor Laboratory, Cold 3. Prescott, D. M. & Kuempel, P. L. (1972) Proc. Natl. Acad. Spring Harbor, NY). Sci. USA 69, 2842-2845. 27. Naito, S., Kitani, T., Ogawa, T., Okazaki, T. & Uchida, H. 4. Sugimoto, K., Oka, A., Sugisaki, H., Takanami, M., Nishi- (1984) Proc. Natl. Acad. Sci. USA 81, 550-554. mura, A., Yasuda, S. & Hirota, Y. (1979) Proc. Natl. Acad. 28. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. Sci. USA 76, 575-579. 29. Fuller, R. S., Bertsch, L. L., Dixon, N. E., Flynn, J. E., Jr., 5. Meijer, M., Beck, E., Hansen, F. G., Bergmans, H. E., Mess- Kaguni, J. M., Low, R. L., Ogawa, T. & Kornberg, A. (1983) er, W., von Meyenburg, K. & Schaller, H. (1979) Proc. Natl. in Mechanisms of DNA Replication and Recombination: Acad. Sci. USA 76, 580-584. UCLA Symposia on Molecular and Cellular Biology, New Se- 6. Oka, A., Sugimoto, K., Takanami, M. & Hirota, Y. (1980) ries, ed. Cozzarelli, N. R. (Liss, New York), Vol. 10, in press. Mol. Gen. Genet. 178, 9-20. 30. Dixon, N. E. & Kornberg, A. (1984) Proc. Natl. Acad. Sci. 7. Asada, K., Sugimoto, K., Oka, A., Takanami, M. & Hirota, Y. USA 81, 424-428. (1982) Nucleic Acids Res. 10, 3745-3754. 31. Burton, K. (1956) Biochem. J. 62, 315-323. 8. Kornberg, A. (1980) DNA Replication (Freeman, San Francis- 32. Maki, H., Horiuchi, T. & Sekiguchi, M. (1983) Proc. Natl. co), pp. 447-448. Acad. Sci. USA 80, 7137-7141. 9. Fuller, R. S. & Kornberg, A. (1983) Proc. Natl. Acad. Sci. 33. Clewell, D. B. (1972) J. Bacteriol. 110, 667-676. USA 80, 5817-5821. 34. Kaguni, J. M., LaVerne, L. S. & Ray, D. S. (1979) Proc. Natl. 10. Fuller, R. S., Kaguni, J. M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 76, 6250-6254. Acad. Sci. USA 78, 7370-7374. 35. Vicuna, R., Hurwitz, J., Wallace, S. & Girard, M. (1977) J. 11. Kaguni, J. M., Fuller, R. S. & Kornberg, A. (1982) Nature Biol. Chem. 252, 2524-2533. (London) 296, 623-627. 36. Hillenbrand, G. & Staudenbauer, W. L. (1982) Nucleic Acids 12. Zyskind, J. W. & Smith, D. W. (1977) J. Bacteriol. 129, 1476- Res. 10, 833-853. 1486. 37. Itoh, T. & Tomizawa, J. (1982) Nucleic Acids Res. 10, 5949- 13. Carl, P. L. (1970) Mol. Gen. Genet. 109, 107-122. 5965. 14. Lark, K. G. (1972) J. Mol. Biol. 64, 47-60. 38. Itoh, T. & Tomizawa, J. (1980) Proc. Natl. Acad. Sci. USA 77, 15. Bagdasarian, M. M., Izakowska, M. & Bagdasarian, M. (1977) 2450-2454. J. Bacteriol. 130, 577-582. 39. Torrey, T. A. & Kogoma, T. (1982) Mol. Gen. Genet. 187, 16. Maal0e, 0. & Hanawalt, P. C. (1961) J. Mol. Biol. 3, 144-155. 225-230. 17. Lark, K. G. & Renger, H. (1969) J. Mol. Biol. 42, 221-225. 40. Kogoma, T., Torrey, T. A. & Connaughton, M. J. (1979) Mol. 18. Kogoma, T. (1978) J. Mol. Biol. 121, 55-69. Gen. Genet. 176, 1-9. 19. Lark, K. G. & Lark, C. A. (1981) in The Initiation of DNA 41. Lark, K. G. & Lark, C. A. (1978) Cold Spring Harbor Symp. Replication, ICN-UCLA Symposia on Molecular and Cellular Quant. Biol. 43, 537-549. Downloaded by guest on September 27, 2021