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Deletion Mutants Are Defective in the Recombination Pathways of Escherichia Coli VEERA M

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Deletion Mutants Are Defective in the Recombination Pathways of Escherichia Coli VEERA M JouRNAL OF BACTERIOLOGY, Aug. 1993, p. 4641-4651 Vol. 175, No. 15 0021-9193/93/154641-11$02.00/0 Copyright C) 1993, American Society for Microbiology Double Helicase II (uvrD)-Helicase IV (helD) Deletion Mutants Are Defective in the Recombination Pathways of Escherichia coli VEERA M. MENDONCA,1 KATHLEEN KAISER-ROGERS,2t AND STEVEN W. MATSON' 2* Department ofBiology' and Curnculum in Geneis,2 University ofNorth Carolina, Chapel Hill North Carolina 27599 Received 16 February 1993/Accepted 30 May 1993 The Escherichia coli helD (encoding helicase IV) and uvrD (encoding helicase H) genes have been deleted, independently and in combination, from the chromosome and replaced with genes encoding antibiotic resistance. Each deletion was verified by Southern blots, and the location of each deletion was confirmed by Pl-mediated transduction. Cell strains containing the single and double deletions were viable, indicating that helicases and IV are not essential for viability. Cell strains lacking helicase IV (&kelD) exhibited no increase in sensitivity to UV irradiation but were slightly more resistant to methyl methanesulfonate (MMS) than the isogenic wild-type cell strain. As expected, cell strains containing the helicase H deletion (AuvrD) were sensitive to both UV irradiation and MMS. The introduction of the helicase IV deletion into a AuvrD background had essentially no effect on the UV and MMS sensitivity ofthe cell strains analyzed. The double deletions, however, conferred a Rec- mutant phenotype for conjugational and transductional recombination in both recBCsbcB(C) and recBC sbcA backgrounds. The Rec- mutant phenotype was more profound in the recBC sbcB(C) background than In the recBC sbc4 background. The recombination-deficient phenotype indicates the direct involvement of helicase and/or helicase IV in the RecF pathway [recBC sbcB(C) background] and RecE pathway (recBC sbc,4 background) of recombination. The modest decrease in the recombination frequency observed in single-deletion mutants in the recBC sbcB(C) background suggests that either helicase is sufficient. In addition, helicase IV has been overexpressed in a tightly regulated system. The data suggest that even modest overexpression of helicase IV is lethal to the cell. DNA helicases, which catalyze the nucleoside triphos- ical activity, there are no genetic data to complement the in phate hydrolysis-dependent unwinding of duplex DNA, play vitro studies and no clear indication as to what role helicase a crucial role in all aspects of DNA metabolism (for reviews, IV plays in the cell. It has been noted that helicase IV shares see references 27 and 28). In Escherichia coli alone, there several physical and biochemical properties with helicase II exist at least 10 distinct helicases which have been purified and Rep protein. In addition, the helicase IV protein con- and characterized (27, 28). A combination of genetic and in tains significant amino acid homology with both helicase II vitro biochemical analyses have implicated these enzymes in and Rep protein (52). In this communication, we report the various facets of nucleic acid metabolism. For example, effect of overexpression of helicase IV and present a partial helicase I participates in F factor-mediated conjugative DNA characterization of its in vivo activity on the basis of genetic transfer (1). DnaB, PriA, and Rep proteins participate in studies of single helD and double helD-uvrD mutants. DNA replication (23, 24, 34). Helicase II, product of the uvrD gene, has been shown to have roles in both methyl- directed mismatch repair and excision repair pathways (30, MATERIALS AND METHODS 32, 37). The latter repair pathway also involves the UvrAB Bacterial strains and plasmids. Bacterial strains, plasmids, complex, which has been demonstrated to have helicase and bacteriophages used in this study are listed in Table 1. activity (31). Finally, largely on the basis of genetic data, the Genotype designations are those of Bachmann (4). All of the different recombination pathways in the cell have been strains involved in recombination and repair assays were shown to utilize the RecBCD enzyme, helicase II, and the derivatives of AB1157 constructed by bacteriophage P1 RecQ helicase (15, 44, 47). Helicase III (53) and helicase IV transduction as described by Miller (29). The identification (51) have yet to be assigned a specific cellular function. of AhelD::cam and AuvrD::tet mutants among the transduc- Helicase IV was identified and purified on the basis of its tants was accomplished by screening for the appropriate DNA unwinding activity and was shown to be a 75-kDa antibiotic resistance, with subsequent analysis of their co- enzyme capable of unwinding duplex DNA in an ATP- transduction frequencies: helD with pyrD (38 to 42%) and dependent reaction (51, 52). The polarity of DNA unwinding uvrD with metE (50%). The helicase II deletions were also is in the 3'-to-5' direction with respect to the bound DNA checked for their sensitivity to UV irradiation. The deletion strand (51). The gene encoding helicase IV, helD, was strains were ultimately confirmed by Southern analysis. The subsequently cloned, sequenced, and mapped to approxi- construction of plasmids pET9d-H4, pT73AH4::cam, and mately 22 min on the E. coli chromosome (52). Since pBSAH2(X) is described below. helicase IV was identified solely on the basis of its biochem- Media and methods. Luria-Bertani medium and M56/2 agar and medium were prepared as previously described (49). Medium was supplemented when required with tetracycline * Corresponding author. (7 pLg/ml), chloramphenicol (25 ,ug/ml), and appropriate t Present address: Department of Pediatrics, University of North amino acids (40 4ug/ml). All other supplements and antibiot- Carolina, Chapel Hill, NC 27599. ics were used at the concentrations recommended by Sam- 4641 4642 MENDONCA ET AL. J. BACTERIOL. TABLE 1. Bacterial strains, plasmids, and bacteriophages Strain designation Significant feature(s) Source, reference, or derivation Cell strains C600 supE44 hsdR thi-1 thr-1 leuB6 lacYl tonA21 rk- mk+ 2 HB101 supE44 hsd2O rB- mB- recA13 aral4proA2 lacYl galK2 rpsL20 7 xyl-S mtl-i RW592 HfrH A(gal-bio) thi Strs su P. Bassford RW592AH2 AuvrD: :tet This study GE1721a pyrD: :cam G. Weinstock GE1752b metE::cam G. Weinstock GE1721AH4 pyrD+ AhelD::cam P1.JC7623AH4 x GE1721 to PyrD+, Camr GE1752AH2 metE+ AuvrD::tet P1.RWAH2 x GE1752 to MetE+, Tet' GEAH2AH4 AuvrD::tet AhelD::cam P1.GEAH4 x GEAH2 to Camr, Tetr HMS174 recA rk]2_ Mk12- Rifr Novagen HMS174(DE3) XDE3 Novagen JC158 HfrPO1 lacI22 X- serA6 spoTi thi-1 10 AB1157c F- thr-1 leuB6 thi-1 lacYl galK2 aral4 xyl5 mtl-i proA2 his4 16 argE3 rpsL3 (Smr) tsx-33 supE44 kdgK51 AB1157 derivativesc AB2463 recA13 17 AB1157AH4 AhelD::cam Pl.GE1721AH4 x AB1157 to Camr AB1157AH2 AuvrD: :tet PL.GE1752AH2 x AB1157 to Tetr AB1157AH2AH4 AhelD::cam AuvrD::tet Pl.GE1721AH4 x ABAH2 to Camr, Tetr JC7623 recB21 recC22 sbcB15 sbcC201 11 JC7623AH4 AhelD::cam This study JC7623AH2 AuvrD::tet Pl.GE1752AH2 x JC7623 to Tet' JC7623AH2AH4 AhelD::cam AuvrD::tet Pl.GE1752AH4 x JC7623AH2 to Camr, Tetr JC8679 recB21 recC22 sbcA23 14 JC8679AH4 AhelD::cam Pl.GE1721AH2 x JC8679 to CaMr JC8679AH2 AuvrD: :tet Pl.GE1752AH2 x JC8679 to Tet' JC8679AH2AH4 AhelD::cam AuvrD::tet PL.GE1752AH4 x JC8679AH2 Cam', Tetr Plasmids pET9d From pBR322, Kan' T7 expression vector Novagen (41) pET9d-H4 Kan', helD contained within a 2.2-kb NcoI-BamHI fragment This study pLysS From pACYC184, CamT encoding T7 lysozyme in the opposite Novagen (41) orientation of the tet promoter pLysE As pLysS, but T7 lysozyme encoded in the same orientation as Novagen (41) the tet promoter pDJ17 AmpT ColEl origin lacZa::cam P. Berget pT73 Ampr ColEl origin 42 pT73H4 Ampr helD contained within a 4.0-kb PstI fragment This study pT73AH4 As pT73H4, but lacks a 1.3-kb SmaI fragment containing the This study helD gene sequence pT73AH4::cam As pT73AH4, but the deleted SmaI fragment has been replaced This study with a 1.4-kb Camr gene pBR322 Ampr Tetr 6 pBluescript M13 Ampr 38 pBSRV.5 As pBluescript, but with uvrD contained within a 5-kb EcoRV This study fragment pGT26 Ampr Tetr uvrD contained within a 2.9-kb PvuII fragment 43 pGT26Atet As pGT26, but Tets This study pGT26AtetAH2 AmpT Tets uvrD flanking sequence contained within a 0.46-kb This study PvuII fragment pGT26AH2::tet Ampr AuvrD::tet contained within a 1.2-kb PvuII fragment This study pTA108 Ampr pSC101 origin 45 pTAH2 As pTA108, but uvrD contained within a 2.9-kb PvuII fragment This study Bacteriophages XCh80del9 4)80 P. Bassford Agtl-XB AattP cI857 C. Joyce kgtl-AH2::tet As Xgtl-XB but AuvrD::tet contained on a 4-kb XbaI fragment This study Xvir VlV2V3 P. Bassford X12G1(553) X2001, with uvrD encoded on a large chromosomal EcoRI 20 fragment a Wild type, except for the pyrD marker. b Wild type, except for the metE marker. C All AB1157 derivatives are isogenic with AB1157, except for the indicated markers. VOL. 175, 1993 HELICASES II AND IV IN RECOMBINATION 4643 brook et al. (33). UV survival and methyl methanesulfonate (MMS) survival assays were performed as previously de- scribed (35). Conjugation experiments for determining re- combination proficiency were performed as described by Willetts et al. (49). Briefly, matings were performed for 1 h at 370C with a 10:1 recipient/donor ratio with cells that had been grown to an optical density at 600 nm (OD6.) of 0.4 (approximately 2 x 108 cells per ml). Viability of Luria- Bertani broth cultures was determined by comparing viable cells (determined by diluting and plating on duplicate Luria- Bertani and M56/2 plates) to the total number of cells (determined by OD6. where an OD6. of 0.4 is approxi- mately 2 x 108 cells per ml).
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