Rep Mutants Research Articles

Charge-to-alanine mutagenesis of the adeno-associated virus type 2 Rep78/68 proteins yields temperature-sensitive and magnesium-dependent variants.

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degrees C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37 degrees C and was undetectable at 39 degrees C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting a ts phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 x 10(6) and 3 x 10(3), respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg(2+)-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep ts mutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.

SbcB sbcC null mutations allow RecF-mediated repair of arrested replication forks in rep recBC mutants.

We have proposed previously that, in Escherichia coli, blockage of replication forks can lead to the reversal of the fork. Annealing of the newly synthesized strands creates a double-stranded end adjacent to a Holliday junction. The junction is migrated away from the DNA end by RuvAB and can be cleaved by RuvC, while RecBCD is required for the repair of the double-stranded tail. Consequently, the rep mutant, in which replication arrests are frequent and fork reversal occurs, requires RecBCD for growth. We show here that the combination of sbcB sbcCD null mutations restores the viability to rep recBC mutants by activation of the RecF pathway of recombination. This shows that the proteins belonging to the RecF pathway are able to process the DNA ends made by the replication fork reversal into a structure that allows recombination-dependent replication restart. However, we confirm that, unlike sbcB null mutations, sbcB15, which suppresses all other recBC mutant defects, does not restore the viability of rep recBC sbcCD strains. We also show that ruvAB inactivation suppresses the lethality and the formation of double-stranded breaks (DSBs) in a rep recBC recF strain, totally deficient for homologous recombination, as well as in rep recBC mutants. This confirms that RuvAB processing of arrested replication forks is independent of the presence of recombination intermediates.

PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling-circle replication.

The only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication for helicase. In contrast, in Bacillus subtilis, in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus. This gene is located at 55 degree on the chromosome and belongs to a putative operon together with a ligase gene (lig) and two unknown genes named pcrB and yerH. As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli. PcrA suppressed the UV sensitivity defect at a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep-phenotype on E. coli. Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.

Isolation of a dnaE mutation which enhances RecA-independent homologous recombination in the Escherichia coli chromosome.

The mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA-independent recombination is increased 15-fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination. It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism. RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.

The Rep78 gene product of adeno-associated virus (AAV) self-associates to form a hexameric complex in the presence of AAV ori sequences.

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences.

The adeno-associated virus (AAV) Rep protein acts as both a repressor and an activator to regulate AAV transcription during a productive infection.

Adeno-associated virus (AAV) uses three promoters, p5, p19, and p40, to regulate viral gene expression. The p5 and p19 promoters direct the synthesis of the viral regulatory proteins, Rep78 and -68 and Rep52 and -40, respectively. The p5 Rep proteins bind a linear 22-bp sequence, the Rep binding element (RBE), that is within both the terminal repeat (TR) and the p5 promoter. In the absence of helper virus, all four Rep proteins have been shown to reduce transcription from the viral p5 and p19 promoters. In this report, we focus on the roles of these proteins and the RBEs in controlling transcription during a productive infection, that is, in the presence of adenovirus. We find that in the presence of adenovirus, the p5 RBE represses p5 transcription while the RBE in the TR activates p5. However, both the TR RBE and the p5 RBE transactivate the p19 and p40 promoters. The fact that the p5 RBE-Rep complex can transactivate p19 and p40 while repressing p5 suggests that Rep78/68 is both a repressor and a transactivator. Rep repression of p5 is specific for the p5 RBE, as other p5 promoter elements do not support this activity. We also demonstrate that in the presence of adenovirus, the p19 Rep proteins, which do not bind to the RBE, can eliminate repression of the p5 promoter by Rep78 and Rep68. This may occur by the association of Rep52 with Rep78 or Rep68 to produce a Rep78/68-Rep52 protein complex which can be detected in vivo by immunoprecipitation. Finally, two Rep mutants that were deficient in RBE binding and transactivation but positive for p5 repression were identified. These mutants may define interaction domains involved in making contacts with other proteins that facilitate repression. These observations suggest a mechanism for controlling the p5 and p19 mRNA levels during a productive AAV infection.

Interaction of wild-type and mutant adeno-associated virus (AAV) Rep proteins on AAV hairpin DNA

Both the Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) bind to AAV terminal repeat hairpin DNA and can mediate site-specific nicking in vitro at the terminal resolution site (trs) within the terminal repeats. To define the regions of the Rep proteins required for these functions, a series of truncated Rep78 derivatives was created. Wild-type and mutant proteins were synthesized by in vitro translation and analyzed for AAV hairpin DNA binding, trs endonuclease activity, and interaction on hairpin DNA. Amino-terminal deletion mutants which lacked the first 29 or 79 amino acid residues of Rep78 did not bind hairpin DNA, which is consistent with our previous identification of a DNA-binding domain in this region. Progressive truncation of the carboxyl-terminal region of Rep78 did not eliminate hairpin DNA binding until the deletion reached amino acid 443. The electrophoretic mobility of the Rep-specific protein-DNA complexes was inversely related to the molecular weight of the Rep derivative. Analysis of the C-terminal deletion mutants by the trs endonuclease assay identified a region (amino acids 467 to 476) that is essential for nicking but is not necessary for DNA binding. When endonuclease-positive, truncated Rep proteins that bound hairpin DNA were mixed with full-length Rep78 or Rep68 protein in electrophoretic mobility shift assays, a smear of protein-DNA complexes was observed. This smear migrated at an intermediate position with respect to the bands generated by the proteins individually. An antibody recognizing only the full-length protein produced a novel supershift band when included in a mixed binding assay containing Rep68 and a truncated Rep mutant. These experiments suggest that the Rep proteins can form hetero-oligomers on the AAV hairpin DNA.

Lethality of rep recB and rep recC double mutants of Escherichia coli.

A rep mutation in combination with a recB or a recC mutation renders Escherichia coli non-viable. This conclusion is based on the following lines of evidence: (i) double mutants cannot be constructed by P1 transduction; (ii) induction of the lambda Gam protein, which inactivates most of the RecBCD activities, is lethal in rep mutants; (iii) rep recBts recCts mutants are not viable at high temperature. The resons for a requirement for the RecBCD enzyme in rep strains were investigated. Initiation of chromosome replication, elongation and chromosomal segregation do not seem impaired in the rep recBtsrecCts mutant at the non-permissive temperature. The viability of other rep derivatives was tested. rep recA recD triple mutants are not viable, whereas rep recD and rep recA double mutants are. Inactivation of both exoV activity and recBC-dependent homologous recombination is therefore responsible for the non-viability of rep recBC strains. However, sbcA and sbcB mutations, which render recBC mutants recombination proficient, do not restore viability of rep recBC mutants, indicating that recombination via the RecF or the RecE pathways cannot functionally replace RecBCD-mediated recombination. The specific requirement for RecBCD suggests the occurrence of double-strand DNA breaks in rep strains. Additional arguments in favour of the presence of DNA lesions in rep mutants are as follows: (i) expression of SOS repair functions delays lethality of rep derivatives after inactivation of RecBCD; (ii) sensitivity of rep strains to ultraviolet light is increased by partial inactivation of RecBCD. A model for the recovery of cells from double-strand breaks in rep mutants is discussed.

Host and phi X 174 mutations affecting the morphogenesis or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate.

The morphogenetic pathway of bacteriophage phi X 174 was investigated in rep mutant hosts that specifically block stage III single-stranded DNA synthesis. The defects conferred by the mutant rep protein most likely affect the formation or stabilization of the 50S complex, a single-stranded DNA synthesizing intermediate, which consists of a viral prohead and a DNA replicating intermediate (preinitiation complex). phi X 174 mutants, ogr (rep), which restore the ability to propagate in the mutant rep hosts, were isolated. The org (rep) mutations confer amino acid substitutions in the viral coat protein, a constituent of the prohead, and the viral A protein, a constituent of the preinitiation complex. Four of the six coat protein substitutions are localized on or near the twofold axis of symmetry in the atomic structure of the mature virion.

Rep protein of tomato yellow leaf curl geminivirus has an ATPase activity required for viral DNA replication.

The Rep protein of geminiviruses is the sole viral protein required for their DNA replication. The amino acid sequence of Rep protein contains an NTP binding consensus motif (P-loop). Here we show that purified Rep protein of tomato yellow leaf curl virus expressed in Escherichia coli exhibits an ATPase activity in vitro. Amino acid exchanges in the P-loop sequence of Rep causes a substantial decrease or loss of the ATPase activity. In vivo, mutant viruses carrying these Rep mutations do not replicate in plant cells. These results show that ATP binding by the Rep protein of geminiviruses is required for its function in viral DNA replication.

Sequence elements of the adeno-associated virus rep gene required for suppressionof herpes-simplex-virus-induced DNA amplification

Herpes simplex virus (HSV) has been shown to induce DNA amplification in the host cell genome, which can be suppressedby the adeno-associated virus type 2 (AAV-2) rep gene (Heilbronn et al., 1990, J. Virol. 64, 3012–3018). In an attempt to define domains of Rep which are required for this effect a set of expression constructs was generated for Rep mutants with either N-terminal and/or C-terminal truncations, with small internal deletions, or with point mutations. In transient cotransfection assays these mutants were tested for the inhibition of HSV-induced DNA amplification and in parallel for DNA replication of a rep-defective AAV genome. Our data show that the C-terminal region of Rep where spliced and unspliced proteins differ is dispensable for both AAV DNA replication and inhibition of HSV-induced DNA amplification. The N-terminus of Rep is required for AAV DNA replication, whereas the first 174 amino acids can be deleted without loss of function for the inhibition of DNA amplification. Rep52 which starts at methionine 225 is neither sufficient, nor required for this effect. We further analyzed the region between amino acids 174 and 225: A stretch of 16 highly hydrophilic amino acids is dispensable for the inhibition of DNA amplification, but it is required for AAV DNA replication. Deletion of two short motifs spanning putative protein kinase C phosphorylation sites each strongly reduce both AAV DNA replication and inhibition of DNA amplification, whereas a single amino acid substitution of one of these sites abolished AAV DNA replication with no effect on the inhibition of DNA amplification. Our data show that most, but not all, of the sequence elements within the N-terminus of Rep78 required for AAV DNA replication coincide with those required for the inhibition of HSV-induced DNA amplification. A replication-negative version of Rep78 comprising the internal 60% of the protein still carry the entire inhibitory function for HSV-induced DNA amplification.

DNA replication studies with coliphage 186: the involvement of the Escherichia coli DnaA protein in 186 replication is indirect

The inability of coliphage 186 to infect productively a dnaA(Ts) mutant at a restrictive temperature was confirmed. However, the requirement by 186 for DnaA is indirect, since 186 can successfully infect suppressed dnaA (null) strains. The block to 186 infection of a dnaA(Ts) strain at a restrictive temperature is at the level of replication but incompletely so, since some 20% of the phage specific replication seen with infection of a dnaA+ host does occur. A mutant screen, to isolate host mutants blocked in 186-specific replication but not in the replication of the close relative coliphage P2, which has no DnaA requirement, yielded a mutant whose locus we mapped to the rep gene. A 186 mutant able to infect this rep mutant was isolated, and the mutation was located in the phage replication initiation endonuclease gene A, suggesting direct interaction between the Rep helicase and phage endonuclease during replication. DNA sequencing indicated a glutamic acid-to-valine change at residue 155 of the 694-residue product of gene A. In the discussion, we speculate that the indirect need of DnaA function is at the level of lagging-strand synthesis in the rolling circle replication of 186.

Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6Kλ origin plasmids at different copy numbers

Escherichia coli hosts were constructed for maintenance of vectors containing the y replication origin of the R6K plasmid ( oriR R6Kλ) at different copy numbers (15 or 250/cell). Such vectors require the trans-acting II protein (the pir gene product) for replication. New hosts carry pir + or pir-116 on the chromosome within uidA, the E. coli gene encoding β-glucuronidase. They were made using the rep technique for allele replacement and Km R M13Δ uidA::pir + or M13Δ uidA::pir-116 phage. Because M13 cannot replicate in a rep mutant, Km R transductants arose by integration into the chromosomal uidA locus. Segregants lacking M13 sequences (which were selected as deoxycholate-resistant (Doc R) ones) frequently contained Δ uidA::pir + or Δ uidA::pir-116 on the chromosome. In principle, this procedure could be used for the introduction of any foreign gene into any nonessential gene on the E. coli chromosome. The Δ uidA::pir + and Δ uidA::pir-116 loci were subsequently transferred to a variety of E. coli strains. One such strain is a suppressor-negative one that is especially useful for transposon (Tn) mutagenesis. This strain has an integrated RP4 derivative for conjugative transfer of oriR R6Kλ plasmids also containing oriT from RP4. In addition, new oriR R6Kλ, oriT + vectors carrying the Tc R−encoding genes tetAR from Tn10 are described. These can be used for allele replacement by conjugative transfer of an oriR R6Kλ, oriT +, tetAR plasmid containing a mutated gene into a non-p(> recipient and by subsequent selection for Tc-sensitive exconjugants.

Construction of new β-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement

Five cassettes carrying uidA, encoding beta-glucuronidase, were made for the construction of insertion mutants with transcriptional fusions to uidA. Three uidA cassettes contain antibiotic-resistance genes, for chloramphenicol (Cm), for kanamycin (Km) and neomycin (Nm), or for streptomycin (Sm) and spectinomycin (Sp). Some cause polar insertions while others provide a promoter for downstream gene expression. The expression of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate use. Several phn::uidA or phn::lacZ insertions were recombined onto the chromosome to test mutational effects and to measure gene expression in single copy. This was done using one of three methods for allele replacement. A new method involved recombination of mutations in M13 onto the chromosome by infection of an Escherichia coli rep mutant that fails to propagate single-stranded DNA phages. Merodiploid recombinants were selected using a resistance marker carried by the M13 phage; segregants lacking M13 sequences were then selected as deoxycholate-resistant (DocR) ones. An improved method for recombination of mutations in pir-dependent, oriR6K vectors involved the use of plasmids containing genes for tetracycline resistance (TcR). Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the pi protein of the R6K plasmid); segregants lacking vector sequences were subsequently selected as Tc-sensitive ones. Both procedures are efficient and allow for recombining marked as well as unmarked mutations onto the chromosome. In addition, some insertions with an antibiotic-resistance marker were directly recombined onto the chromosome by transformation of a recD mutant with linear DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Partition of Plasmid R1: A Computer Simulation

A computer-simulated population of individual Escherichia coli cells harboring plasmid R1 parA +/parB - has been used to analyze three possible modes of plasmid segregation: equipartition, in which plasmids are partitioned equally to daughter cells at cell division; single-site inheritance, in which the products of the most recent replication event are partitioned equally and the remaining plasmids are distributed randomly; and pair-site partition, in which a single, randomly chosen plasmid is actively partitioned to each daughter cell at division and the rest are distributed randomly. Comparison between predicted and experimental plasmid loss-frequency enabled us to rule out the first of these models as a likely mode of action in R1 but was inconclusive regarding the other two. The parA region would therefore seem to partition actively only one pair of plasmids to each daughter cell, the precise selection rule involved remaining unresolved. This question is not easily decided with current technology, as we show, but our simulation results also predict that the isolation of rep (Ts) mutants will provide an experimental system in which a clear distinction is possible between two plasmids that are the products of the most recent replication event and two that are chosen strictly at random.

Sequence determination and characterization of the replicator region in the tumor-inducing plasmid pTiB6S3

The replicator region of the 195-kilobase-pair (kb) tumor-inducing plasmid pTiB6S3 was previously identified by isolation of a 6.8-kb miniplasmid (B.P. Koekman, P.J.J. Hooykaas, and R.A. Schilperoort, Plasmid 7:119-132, 1982). This miniplasmid was joined to ColE1-based vectors and subjected to mutagenesis. The resulting mutant plasmids were examined for their ability to replicate autonomously in Agrobacterium tumefaciens. It was found that a 4.2-kb region was sufficient for displaying replication characteristics similar to those of the parental pTiB6S3. Nucleotide sequence analysis of this 4.2-kb region revealed the presence of three possible reading frames in the same direction (repA, repB, and repC). Proteins coded for by these frames were identified by in vitro synthesis in a coupled transcription-translation system. The replicating ability became attenuated by repA and repB mutations but was completely abolished by repC mutations. The size, arrangement, and mutational effects of the three rep genes were quite similar to those of the rep genes that were previously identified in the hairy root-inducing plasmid pRiA4b. However, defects caused by rep mutations in one plasmid were unable to be complemented by corresponding functions in the other plasmid.

The Escherichia coli rep mutation. X. Consequences of increased and decreased Rep protein levels.

Recombinant DNA techniques were used to study various aspects of rep gene function in Escherichia coli. In order to enhance expression of the Rep protein, the rep gene was cloned into the vector pKC30 under the control of the lambda pL promoter. By trimming away a portion of the DNA sequence immediately upstream of the translational start site of rep, we were able to obtain very high levels of Rep protein upon induction. Cells carrying such plasmids showed no ill effects from the high concentration of the protein. To ascertain the consequence of the absence of Rep protein on the cell, the chromosomal copy of the gene was deleted using a homologous recombination technique. The viability of E. coli strains completely lacking the rep gene proves that the Rep function is not essential, at least in wild-type cells under laboratory conditions. We confirmed that in the absence of Rep function there is an increase in the average number of growing forks in exponentially growing cells; augmentation of Rep protein levels above normal, however, did not detectably decrease the number of growing forks.

Comparison of the rep-38 and mmrA1 mutations of Escherichia coli

The rep-38 and mmrA1 mutations are located very close to each other (∼85 min), and have been suggested to be allelic. To address this question we have compared the phenotypes of the mmrA1 and rep-38 mutants. Both the mmrA1 and rep-38 mutations blocked the enhanced killing and inhibition of postreplication repair by rich growth medium that occurs in UV-irradiated Escherichia coli K-12 uvrA cells, i.e., the mmrA1 and rep-38 strains did not show minimal medium recovery (MMR). However, ΦX174 bacteriophage propagated well in mmrA1 strains, but not in rep-38 strains; a rep mutation sensitized a uvrA strain to UV irradiation, but a mmrA mutation did not. During chloramphenicol treatment, the rep-38 strain showed a larger amount of residual DNA synthesis than observed in the mmrA1 strain. The mmrA1 mutation appears to be a dominant mutation. This was determined by the failure of either plasmid pLC44-7 or episome F′KLF11, both of which carry the mmrA + gene, to complement the Mmr − phenotype of a uvrA mmrA strain. Plasmid pLC44-7 is known to complement the rep-38 mutation, suggesting that rep-38 is a recessive mutation. Although certain of the phenotypes of the rep and mmrA mutants are similar, a number are quite different. These differences suggest that these two mutations are not allelic.

M13-oriC cloning vehicles: use for amplification of high copy lethal (HCL) genetic elements.

M13 cloning vehicles have been constructed which contain the Escherichia coli origin for DNA replication (oriC), with and without selectable antibiotic-resistance genes. Since the M13 viral strand origin requires a functional rep gene product, using oriC these vehicles propagate as low-copy-number plasmids in E. coli rep mutants. This property is exploited to amplify cloned "high copy lethal" (HCL) DNA fragments, those containing genetic elements which kill the E. coli host when present at multiple copies in the cell. Following cloning of such fragments in these vehicles and initial selection in E. coli rep cells, the M13-oriC chimeric plasmid DNA is used to transfect appropriate E. coli rep+ cells. The chimeric DNA propagates as M13 viral DNA, yielding double-stranded and single-stranded DNA products and phage particles prior to killing of the host via expression of the HCL element; these events mimic a lytic phage infection. Such amplification will greatly facilitate both DNA "library" constructions (HCL elements are absent a priori from libraries using high-copy-number cloning vehicles) and studies of HCL elements including restriction mapping, DNA sequencing, and physiological studies.

Lethality of the double mutations rho rep and rho ssb in Escherichia coli.

The similarity of rho mutants to rep and ssb mutants in sensitivity to UV light and in recombination deficiency suggested that the function of the Rho protein might be related to that of Rep and Ssb. In support of that idea, we found that rho rep and rho ssb double mutants are either nonviable, or at best only marginally viable. Viability could be restored by suppressor mutations, one of which mapped either in the rho gene or close to its 5'-end. Rho may thus share a role with Rep and Ssb in replication and the structural maintenance of DNA; a multifunctional Rho protein could account for the diversity of the defects seen in rho mutants, some of which appear to have no relation to the defect in transcription termination.