Kinetics of binding of single-stranded DNA binding protein from Escherichia coli to single-stranded nuclei acids

The time course of the reaction of Escherichia coli single-stranded DNA binding protein (E. coli SSB) with poly(dT) and M13mp8 single-stranded DNA has been measured by fluorescence stopped-flow experiments. For poly(dT), the fluorescence traces follow simple bimolecular behavior up to 80% saturation of the polymer with E. coli SSB. A mechanistic explanation of this binding behavior can be given as follows: (1) E. coli SSB is able to translocate very rapidly on the polymer, forming cooperative clusters. (2) In the rate-limiting step of the association reaction, E. coli SSB is bound to the polymer only by one or two of its four contact sites. As compared to poly(dT), association to single-stranded M13mp8 phage DNA is slower by at least 2 orders of magnitude. We attribute this finding to the presence of secondary structure elements (double-stranded structures) in the natural single-stranded DNA. These structures cannot be broken by E. coli SSB in a fast reaction. In order to fulfill its physiological function in reasonable time, E. coli SSB must bind newly formed single-stranded DNA immediately. The protein can, however, bind to such pieces of the newly formed single-stranded DNA which are too short to cover all four binding sites of the E. coli SSB tetramer.     

Contrasting effects of Escherichia coli single-stranded DNA binding protein on synthesis by T7 DNA polymerase and Escherichia coli DNA polymerase I (large fragment). Evidence that binding protein inhibits trans-lesion synthesis by polymerase I

The effect of Escherichia coli single-stranded DNA binding protein (SSB) on DNA synthesis by T7 DNA polymerase and E. coli DNA polymerase I (large fragment) using native or aminofluorene-modified M13 templates was evaluated by in vitro DNA synthesis assays and polyacrylamide gel electrophoresis analysis. The two polymerase enzymes displayed differential responses to the addition of SSB. T7 DNA polymerase, a enzyme required for the replication of the T7 chromosome, was stimulated by the addition of SSB whether native or modified templates were used. On the other hand, E. coli DNA polymerase I was slightly stimulated by the addition of SSB to the native template but substantially inhibited on modified templates. This result suggests that DNA polymerase I may be able to synthesize past an aminofluorene adduct but that the presence of SSB inhibited this trans-lesion synthesis. Polyacrylamide gels of the products of DNA synthesis by polymerase I supported this inference since SSB caused a substantial increase in the accumulation of shorter DNA chains induced by blockage at the aminofluorene adduct sites.     

Repair of thymine.guanine and uracil.guanine mismatched base-pairs in bacteriophage M13mp18 DNA heteroduplexes

Repair of thymine.guanine (T.G) and uracil.guanine (U.G) mismatched base-pairs in bacteriophage M13mp18 replicative form (RF) DNA was compared upon transfection into repair-proficient or repair-deficient Escherichia coli strains. Oligonucleotide-directed mutagenesis was used to prepare covalently closed circular heteroduplexes that contained the mismatched base-pair at a restriction recognition site. The heteroduplexes were unmethylated at dam (5'-GATC-3') sites to avoid methylation-directed biasing of repair. In an E. coli host containing uracil-DNA glycosylase (ung+), about 97% of the transfecting U.G-containing heteroduplexes had the U residue excised by the uracil-excision repair system. With the analogous T.G mispair, mismatch repair operated on almost all of the transfecting heteroduplexes and removed the T residue in about 75% of them when the mismatched T was on the minus strand of the RF DNA. Similar preferential excision of the minus-strand's mismatched base was observed whether the heteroduplex RF DNA molecules had only one or both strands unmethylated at dcm (5'-CC(A/T)GG-3') sites and whether the RF DNA was prepared by primer extension in vitro or by reannealing mutant and non-mutant DNA strands. Also, the extent and directionality of repair was the same at a U.G mispair in ung- host cells as at the analogous T.G mispair in ung- or ung+ cells. Only in a mismatch repair-deficient (mutH-) host was the plus strand of the transfecting M13mp18 heteroduplex DNA preferentially repaired. It is suggested that the plus strand nick made by the M13-encoded gene II protein might be employed by a mutH- host to initiate repair on that strand.     

Single-stranded DNA binding protein from calf thymus. Purification, properties, and stimulation of the homologous DNA-polymerase-alpha-primase complex.

A binding protein for single-stranded DNA (ssDNA) was purified from calf thymus to near homogeneity by chromatography on DEAE-cellulose, blue-Sepharose, ssDNA-cellulose and FPLC Mono Q. The most purified fraction consisted of four polypeptides with molecular masses of 70, 55, 30, and 11 kDa. The polypeptide with the molecular mass of 55 kDa is most likely a degraded form of the largest polypeptide. The complex migrated as a whole on both glycerol gradient ultracentrifugation (s = 5.1 S) and gel filtration (Stokes' radius approximately 5.1 nm). Combining these data indicates a native molecular mass of about 110 kDa, which is in accord with a 1:1:1 stoichiometry for the 70 + 55/30/11-kDa complex. The ssDNA binding protein (SSB) covered approximately 20-25 nucleotides on M13mp8 ssDNA, as revealed from both band shift experiments and DNase I digestion studies. The homologous DNA-polymerase-alpha-primase complex was stimulated by the ssDNA binding protein 1.2-fold on poly(dA).(dT)14 and 10-13-fold on singly primed M13mp8 DNA. Stimulation was mainly due to facilitated DNA synthesis through stable secondary structures, as demonstrated by the vanishing of many, but not all, pausing sites. Processivity of polymerase-primase was not affected on poly(dA).(dT)14; with poly(dT).(rA)10 an approximately twofold increase in product lengths was observed when SSB was present. The increase was attributed to a facilitated rebinding of polymerase alpha to an already finished DNA fragment rather than to an enhancement of the intrinsic processivity of the polymerase. Similarly, products 300-600 nucleotides long were formed on singly primed M13 DNA in the presence of SSB, in contrast to 20-120 nucleotides when SSB was absent. DNA-primase-initiated DNA replication on M13 DNA was inhibited by SSB in a concentration-dependent manner. However, with less sites available to begin with RNA priming, more homogeneous products were formed.     

Selective reactivities of isocyanates towards DNA bases and genotoxicity of methylcarbamoylation of DNA.

The reactivities of methyl isocyanate (MIC) and phenyl isocyanate (PIC) with DNA, and the genotoxicity of MIC were investigated. MIC and PIC reacted with the exocyclic amino group of deoxycytidine, deoxyadenosine and deoxyguanosine to produce carbamoylated products. The reactions of both isocyanates with deoxycytidine were 2 and 4 orders of magnitude higher than with deoxyadenosine and deoxyguanosine, respectively. To explore the genotoxicity of MIC, M13mp9 RF DNA was modified with MIC and then introduced into E. coli. The plaque-forming efficiencies of DNA decreased with increasing dose levels, and the decreases were more pronounced in Uvr endonuclease-deficient strains (uvrA, uvrB and uvrC) than in the Uvr endonuclease-proficient strain, JM103. The differences in survival in JM103 and uvr- strains suggest that the methylcarbonyl adducts can be removed by the uvr excision-repair system. Modification of M13mp9 RF DNA with MIC induced MIC-dose-related, SOS-dependent mutations in the beta-galactosidase locus. These results demonstrate the genotoxic response of MIC-modified DNA in E. coli.     

Biological properties of imidazole ring-opened N7-methylguanine in M13mp18 phage DNA.

Guanine residues methylated at the N-7 position (7-MeGua) are susceptible to cleavage of the imidazole ring yielding 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7-MeGua). The presence of Fapy-7-MeGua in DNA template causes stops in DNA synthesis in vitro by E. coli DNA polymerase I. The biological consequences of Fapy-7-MeGua lesions for survival and mutagenesis were investigated using single-stranded M13mp18 phage DNA. Fapy-7-MeGua lesions were generated in vitro in phage DNA by dimethylsulfate (DMS) methylation and subsequent ring opening of 7-MeGua by treatment with NaOH (DMS-base). The presence of Fapy-7-MeGua residues in M13 phage DNA correlated with a significant decrease in transfection efficiency and an increase in mutation frequency in the lacZ gene, when transfected into SOS-induced JM105 E.coli cells. Sequencing analysis revealed unexpectedly, that mutation rate at guanine sites was only slightly increased, suggesting that Fapy-7-MeGua was not responsible for the overall increase in the mutagenic frequency of DMS-base treated DNA. In contrast, mutation frequency at adenine sites yielding A----G transitions was the most frequent event, 60-fold increased over DMS induced mutations. These results show that treatment with alkali of methylated single-stranded DNA generates a mutagenic adenine derivative, which mispairs with cytosine in SOS induced bacteria. The results also imply that the Fapy-7-MeGua in E. coli cells is primarily a lethal lesion.     

Mechanism of stimulation of T7 DNA polymerase by Escherichia coli single-stranded DNA binding protein (SSB)

Single-stranded DNA binding protein is a key component in growth of bacteriophage T7. In addition, DNA synthesis by the purified in vitro replication system is markedly stimulated when the DNA template is coated with Escherichia coli single-stranded DNA binding protein (SSB). In an attempt to understand the mechanism for this stimulation, we have studied the effect of E. coli SSB on DNA synthesis by the T7 DNA polymerase using a primed single-stranded M13 DNA template which serves as a model for T7 lagging strand DNA synthesis. Polyacrylamide gel analysis of the DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase pauses at many specific sites, some stronger than others. By comparing the position of pausing with the DNA sequence of this region and by using a DNA template that contains an extremely stable hairpin structure, it was found that many, but not all, of these pause positions correspond to regions of potential secondary structure. The presence of SSB during synthesis resulted in a large reduction in the frequency of hesitations at many sites that correspond to these secondary structures. However, the facts that a large percentage of the pause sites remain unaffected even at saturating levels of SSB and that SSB stimulates synthesis on a singly primed poly(dA) template suggested that other mechanisms also contribute to the stimulation of DNA synthesis caused by SSB. Using a sucrose gradient analysis, we found that SSB increases the affinity of the polymerase for single-stranded DNA that this increased binding is only noticed when the polymerase concentration is limiting. The effect of this difference in polymerase affinity was clearly observed by a polyacrylamide gel analysis of the product DNA synthesized during a limited DNA synthesis reaction using conditions where only two nucleotides are added to the primer. Under these circumstances, where the presence of hairpin structures should not contribute to the stimulatory effect of SSB, we found that the extension of the primer is stimulated 4-fold if the DNA template is coated with SSB. Furthermore, SSB had no effect on this synthesis at large polymerase to template ratios.     

Site-specific mutagenesis in vivo by single methylated or deaminated purine bases

The technique of site-directed mutagenesis has been used to investigate the mutagenicity of O6-methylguanine (O6-MeG) or hypoxanthine introduced as a single lesion at a specific locus in an M13mp9 RF molecule constructed in vitro. Following transformation of O6-MeG-containing RF molecules into E. coli JM101, mutant progeny phage were produced at a frequency not significantly different from that observed with wild-type M13mp9 RF. The mutant yield was greatly enhanced by exhausting cellular O6-MeG DNA-methyltransferase before transformation. In contrast, hypoxanthine exhibited miscoding mutagenesis in the absence of interference with cellular repair mechanisms. This indicates that cellular hypoxanthine-DNA glycosylase acts inefficiently in the removal of hypoxanthine from DNA in vivo. The precise mutational changes induced by hypoxanthine were determined by DNA sequence analysis.     

Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct

The ability of Escherichia coli DNA polymerase I and T7 DNA polymerase to bypass bulky C-8 guanyl-2-aminofluorene adducts in DNA was studied by in vitro DNA synthesis reactions on a site-specific aminofluorene-modified M13mp9 template. This site-specifically modified DNA was prepared by ligating an oligonucleotide containing a single aminofluorene adduct into a gapped heteroduplex of M13mp9 DNA (Johnson, D. L., Reid, T. M., Lee, M.-S., King, C. M., and Romano, L. J. (1986) Biochemistry 25, 449-456). The resulting covalently closed duplex DNA molecule was then cleaved with a restriction endonuclease, denatured, and annealed to a primer on the 3' side of the adduct to form a template specifically designed to study bypass. In this system, any synthesis that was not blocked by the bulky aminofluorene adduct would proceed to the 5' terminus of the single-stranded template, while synthesis interrupted by the adduct would terminate at or near the adduct location. We have measured DNA synthesis on this template and find that the amount of radiolabeled nucleotide incorporated by either E. coli DNA polymerase I (large fragment) or T7 DNA polymerase was much greater than would be predicted if the aminofluorene adduct were an absolute block to DNA synthesis. Furthermore, the products of similar reactions electrophoresed on polyacrylamide gels showed conclusively that the majority of the DNA synthesized by either the T7 DNA polymerase or E. coli DNA polymerase I bypassed the aminofluorene lesion. Substitution of Mn2+ for Mg2+ as the divalent cation resulted in even higher levels of translesion synthesis.     

Effect of synthetic hydroxy isothiocyanates on a bacterial virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on psiK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Effect of Synthetic Hydroxy Isothiocyanates on a Bacterial Virus and DNA

The Effect of hydroxy isothiocyanates on a bacterial virus and M13 DNA was examined. Hydroxy-substituted phenyl and phenyl alkyl isothiocyanates, especially 2-(3,4-dihydroxyphenyl)ethyl isothiocyanate(IT-Dop) synthesized from dopamine, showed antiviral activity on φK. In transfection experiments with M13 mp DNA species, IT-Dop inhibited the single-stranded (SS) molecule more effectively than the double stranded replicative form (RF) DNA. These effects were dependent on reaction time, and on IT-Dop concentration. An additional experiment indicated that treatment with IT-Dop suppressed annealing (reassociation) of denatured DNA. These results indicate that IT-Dop reacts mildly with virus and SS DNA.     

Effects of the Escherichia coli SSB protein on the binding of Escherichia coli RecA protein to single-stranded DNA. Demonstration of competitive binding and the lack of a specific protein-protein interaction

The effect of the Escherichia coli single-stranded DNA binding (SSB) protein on the stability of complexes of E. coli RecA protein with single-stranded DNA has been investigated through direct DNA binding experiments. The effect of each protein on the binding of the other to single-stranded DNA, and the effect of SSB protein on the transfer rate of RecA protein from one single-stranded DNA molecule to another, were studied. The binding of SSB protein and RecA protein to single-stranded phage M13 DNA is found to be competitive and, therefore, mutually exclusive. In the absence of a nucleotide cofactor, SSB protein binds more tightly to single-stranded DNA than does RecA protein, whereas in the presence of ATP-gamma-S, RecA protein binds more tightly than SSB protein. In the presence of ATP, an intermediate result is obtained that depends on the type of DNA used, the temperature, and the magnesium ion concentration. When complexes of RecA protein, SSB protein and single-stranded M13 DNA are formed under conditions of slight molar excess of single-stranded DNA, no effect of RecA protein on the equilibrium stability of the SSB protein-single-stranded DNA complex is observed. Under similar conditions, SSB protein has no observed effect on the stability of the RecA protein-etheno M13 DNA complex. Finally, measurements of the rate of RecA protein transfer from RecA protein-single-stranded DNA complexes to competing single-stranded DNA show that there is no kinetic stabilization of the RecA protein-etheno M13 DNA complex by SSB protein, but that a tenfold stabilization is observed when single-stranded M13 DNA is used to form the complex. However, this apparent stabilizing effect of SSB protein can be mimicked by pre-incubation of the RecA protein-single-stranded M13 DNA complex in low magnesium ion concentration, suggesting that this effect of SSB protein is indirect and is mediated through changes in the secondary structure of the DNA. Since no direct effect of SSB protein is observed on either the equilibrium or dissociation properties of the RecA protein-single-stranded DNA complex, it is concluded that the likely effect of SSB protein in the strand assimilation reaction is on a slow step in the association of RecA protein with single-stranded DNA. Direct evidence for this conclusion is presented in the accompanying paper.     

Use of M13mp phages to study gene regulation, structure and function: cloning and recombinational analysis of genes of the Salmonella typhimurium histidine operon

A restriction map was determined for a phi 80 lambda dhis transducing phage DNA carrying the Salmonella typhimurium histidine operon. DNA fragments containing the promoter/regulatory region and the first two structural genes of the histidine operon (hisOGD) were identified by their ability to direct regulated synthesis of histidinol dehydrogenase (product of hisD) in a coupled in vitro protein synthesizing system. A 3.1-kb SalI-EcoRI restriction fragment containing the hisOGD region, was subcloned into phage M13mp8 and M13mp9 RF DNAs. Methods are described for shuttling mutant and wild-type bacterial DNA sequences between the M13mp::his phage and host bacterial genomes. Of novel importance is the use of the phage M13 gene II amber mutation to obtain integration of the M13mp::his phage genome into the homologous his region of the bacterial chromosome following transduction of recipients lacking an amber suppressor. This method can be used to facilitate allele replacement with genes carried on M13 transducing phages.     

Promotion of double-strand break repair by human nuclear extracts preferentially involves recombination with intact homologous DNA.

Parameters of DNA double strand break (dsb) repair catalysed by human nuclear extract were analysed using, as substrate, the replicative form (RF) of M13 mp8 in which a single double strand break (dsb) was introduced by restriction. After incubation with the extract, the dsb repair was estimated by the ability of the incubated RF to produce plaques following transfection into JM 109 (Rec A-) bacteria. The possibility of recombination with a purified fragment from M13 mp8 RF enhances up to 20 times the plaquing ability of the RF. The repair by recombination occurs under several conditions: i) the break in the RF must be located in the region of homology with the fragment. ii) the fragment has to be intact in the region corresponding to the break in the RF. iii) a minimal length of homology between the region surrounding the dsb in the RF, and the fragment is required. The in vitro reaction is ATP dependent and dNTP's partially dependent. Dephosphorylation of the free ends in the RF decreases the repair by ligation but is without effect on the recombination.     

Singlet oxygen-induced mutations in M13 lacZ phage DNA

The mutagenic consequences of damages to M13 mp19 RF DNA produced by singlet oxygen have been determined in a forward mutational system capable of detecting all classes of mutagenic events. When the damaged M13 mp19 RF DNA is used to transfect competent E. coli JM105 cells, a 16.6-fold increase in mutation frequency is observed at 5% survivors when measured as a loss of alpha-complementation. The enhanced mutagenicity is largely due to single-nucleotide substitutions, frameshift events and double-mutations. The single-nucleotide substitutions occur in the regulatory and in the structural part of the lacZ gene under the predominant form of a G:C to T:A transversion. The spectrum of mutations detected among the M13 lacZ phages surviving the singlet oxygen treatment is totally different from those appearing spontaneously. SOS induction mediated through u.v.-irradiation of bacteria leads to an increase of the mutation frequency in the M13 surviving to the singlet oxygen treatment. The mutation spectrum in this case is a mixture between those observed with the spontaneous mutants and the mutants induced by singlet oxygen. Lesions introduced in the M13 mp19 RF DNA can be partly repaired by the enzymatic machinery of the bacteria. It turns out that excision-repair and SOS repair are probably involved in the removal of these lesions by singlet oxygen.     

Mechanism of stimulation of DNA replication by bacteriophage phi 29 single-stranded DNA-binding protein p5

Protein p5 is a Bacillus subtilis phage phi 29-encoded protein required for phi 29 DNA replication in vivo. Protein p5 has single-stranded DNA binding (SSB) capacity and stimulates in vitro DNA replication severalfold when phi 29 DNA polymerase is used to replicate either the natural phi 29 DNA template or primed M13 single-stranded DNA (ssDNA). Furthermore, other SSB proteins, including Escherichia coli SSB, T4 gp32, adenovirus DNA-binding protein, and human replication factor A, can functionally substitute for protein p5. The stimulatory effect of phi 29 protein p5 is not due to an increase of the DNA replication rate. When both phi 29 DNA template and M13 competitor ssDNA are added simultaneously to the replication reaction, phi 29 DNA replication is strongly inhibited. This inhibition is fully overcome by adding protein p5, suggesting that protein p5-coated M13 ssDNA is no longer able to compete for replication factors, probably phi 29 DNA polymerase, which has a strong affinity for ssDNA. Electron microscopy demonstrates that protein p5 binds to M13 ssDNA forming saturated complexes with a smoothly contoured appearance and producing a 2-fold reduction of the DNA length. Protein p5 also binds to ssDNA in the phi 29 replicative intermediates produced in vitro, which are similar in structure to those observed in vivo. Our results strongly suggest that phi 29 protein p5 is the phi 29 SSB protein active during phi 29 DNA replication.     

Inhibition of bacteriophage M13 and phix174 duplex DNA replication and single-strand synthesis in temperature-sensitive dnaZ mutants of Escherichia coli.

A functional dnaZ product, known to be essential for host DNA polymerization and for the synthesis of M13 and phiX174 parental replicative-form (RF) DNA, is required also for RF replication and single-strand synthesis by both of these phages. All three stages of M13 and phiX174DNA replication (parental RF formation, RF replication, and single-strand synthesis) are inhibited in dnazts mutants at elevated temperatures. In addition, the thermolabile step in M13 parental RF formation appears to occur after RNA priming;i.e., the synthesis of M13 RF DNA proceeded when a dnaZts mutant, infected at a nonpermissive temperature, was transferred to a permissive temperature in the presence of rifampin.     

Bisulfite-induced cytosine deamination rates in E. coli SSB:DNA complexes

E. coli single-stranded binding protein (SSB) has been examined for its ability to modulate bisulfite-induced cytosine deamination rates in single-stranded DNA (ssDNA). We used a lacZ alpha-complementation reversion assay to detect C-->U rates at a single codon in M13mp2 DNA, whether in free ssDNA or in an SSB:ssDNA complex. When incubated at 37 degrees C, the average bisulfite-induced reversion rate constant was four-fold less in SSB:ssDNA complexes than in ssDNA, at a single codon. Across a 250 base pair target and over 23 scorable C-->U sites, the forward rate constant was 4.9-fold less in SSB:ssDNA complexes than in ssDNA alone. After treatment with N-uracil glycosylase, ssDNA incubated with bisulfite had reversion frequencies at the background rate of ssDNA incubated without bisulfite, indicating that virtually all mutations scored were due to C-->U events. The decrease in cytosine deamination rates occurred both in a single codon and over a 250 bp target, indicating that interactions between SSB and ssDNA reduce bisulfite-catalyzed mutations. The structural role of SSB is well recognized in multiple cellular processes; SSB can also function to minimize bisulfite-induced ssDNA mutations.     

SOS-independent mutagenesis in lacZ induced by methylene blue plus visible light

Summary In vitro photosensitization by visible light in the presence of methylene blue (MB-light) produces lesions in M13mpl8 lacZ phage DNA, the lethal and mutagenic potential of which was analyzed after transfection into various bacterial hosts. Mutagenesis was determined with a forward mutation assay using the lacZ gene of M13mp18 as a target. When, MB-light-treated double-stranded (ds) M13mp18 DNA was used to transfect wild-type cells which were not induced for SOS functions, a fivefold increase in mutation frequency was observed at 10% survival compared to that observed with untreated DNA. Mutation frequency obtained with MB-light-treated ds M13mp18 DNA was greater when transfected into the uvrA fpg-1 double mutant than that seen in uvrA, fpg-1, or umuC single mutants or in the wild-type. Sequence analysis shows that in the wild-type strain, MB-light treatment of ds M13mp18 DNA results mostly in single base substitutions. The most frequent base change is the GCTA transversion. MB-light treatment of single-stranded (ss) M13mp18 DNA also results in an increased mutation frequency after transfection into the wild-type strain, yielding mostly GT transversions. Our results show that MB-light-induced mutagenesis is at least partially independent of the induction of SOS functions in Escherichia coli. The mutation spectra suggest that 8-oxo-7,8-dihydroguanine is the major promutagenic lesion in DNA.     

Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors

Three kinds of improvements have been introduced into the M13-based cloning systems. (1) New Escherichia coli host strains have been constructed for the E. coli bacteriophage M13 and the high-copy-number pUC-plasmid cloning vectors. Mutations introduced into these strains improve cloning of unmodified DNA and of repetitive sequences. A new suppressorless strain facilitates the cloning of selected recombinants. (2) The complete nucleotide sequences of the M13mp and pUC vectors have been compiled from a number of sources, including the sequencing of selected segments. The M13mp18 sequence is revised to include the G-to-T substitution in its gene II at position 6 125 bp (in M13) or 6967 bp in M13mp18. (3) M13 clones suitable for sequencing have been obtained by a new method of generating unidirectional progressive deletions from the polycloning site using exonucleases HI and VII.