Cloning and delimiting one chloroplast DNA replicative origin of Chlamydomonas.

The EcoR1 restriction fragments containing D-loops which marked the replication origin of chloroplast DNA were identified in two different species of Chlamydomonas. Each fragment was cloned in the E. coli plasmid pBR325. The cloned fragments were compared by restriction endonuclease analyses and by heteroduplex analyses in the electron microscope. The relative position of the D-loop regions and the homologous regions between the 2 fragments was determined. The D-loops were located within one short homologous region of 0. 42kb in length between the 2 cloned restriction fragments. The homologous region was subcloned in pBR322. Closed circular plasmid DNAs containing the short homologous region showed preferred denaturation in the D-loop region under physiological salt concentration which suggested that D-loop region was AT rich. Sequence divergence was detected at both ends of the D-loop region. Southern blot analyses indicated the presence of species-specific repetitive sequences within the divergent regions.     

In vitro sodium bisulfite mutagenesis of restriction endonuclease recognition sites

Sodium bisulfite treatment of single-stranded DNA deaminates exposed cytosine residues to form uracil, resulting in cytosine-to-thymidine transition mutations following DNA replication. We have used this reaction in vitro to destroy the recognition sequences for the restriction endonucleases HindIII and XmaI in the aminoglycoside 3'-phosphotransferase I coding region of plasmid pUC4K. This procedure should be applicable to the mutation of any recognition sequence of restriction endonucleases which generate cytosine-containing single-stranded ends. The possibility of mutagenesis of restriction sites to generate stop codons in coding regions is discussed.     

The structure of the initiation complex at the replication origin, oriC, of Escherichia coli.

Two distinct regions in the replication origin, oriC, of Escherichia coli are separately distorted upon initiation complex formation by the initiator protein DnaA. The AT-rich region in the left part of oriC and the start site region in the right part of oriC. Chemical modification of single-stranded DNA was observed at both regions whereas endonuclease recognition of DNA mini-bulges specifically occurred in the start site region. We show that the helical phasing of binding sites for DnaA protein in oriC is important for origin function. An insertion or deletion of one helical turn between the two rightmost binding sites does not alter the efficiency of replication initiation, whereas all modifications of distance by less or more than one helical turn result in inactivation of oriC. DnaA binding and helical distortions in the AT-rich region as well as in the start site region are not affected in the distance mutants irrespective of their functionality in vivo. We propose a specific compact nucleoprotein structure for the initiation complex.     

Analysis of single-stranded DNA stability and damage-induced strand loss in mammalian cells using SV40-based shuttle vectors

The fate and stability of fully or partially single-stranded DNA molecules transfected into mammalian cells have been analysed. For this, we constructed a simian virus 40 (SV40)-based shuttle vector containing the f1 bacteriophage replication origin in the two possible orientations (pi SVF1-A and pi SVF1-B). This vector contains the SV40 origin of replication, the late viral genes and DNA sequences for replication and selection in Escherichia coli. It also carries the lacO sequence, which permits the analysis of plasmid stability. Single-stranded DNA from pi SVF1-A and pi SVF1-B were produced in bacteria and annealed in vitro to form a heteroduplex molecule. We showed that, in monkey kidney COS7 cells, single-stranded vectors replicate to form duplex molecules. After transfection of the three forms of molecules (single-stranded, heteroduplex or double-stranded), replicated DNA was rescued in E. coli. Vector stability was analysed by checking for plasmid rearrangements and screening for lacO mutants. The single-stranded pi SVF1 has a lower rearrangement level, while the spontaneous mutation frequency (on the lacO target) is in the same range as for the double-stranded vector. In contrast, the level of spontaneous mutagenesis is higher for the heteroduplex than for the single- and double-stranded forms. In addition, we found that replication of heteroduplex with one strand containing ultraviolet light-induced lesions yields progeny molecules in which the irradiated strand is mostly lost. This result indicates for the first time the specific loss of the damaged strand in mammalian cells.     

Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin

The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.     

Localized DNA melting and structural pertubations in the origin of replication, oriC, of Escherichia coli in vitro and in vivo.

The leftmost region of the Escherichia coli origin of DNA replication (oriC) contains three tandemly repeated AT-rich 13mers which have been shown to become single-stranded during the early stages of initiation in vitro. Melting is induced by the ATP form of DnaA, the initiator protein of DNA replication. KMnO4 was used to probe for single-stranded regions and altered DNA conformation during the initiation of DNA replication at oriC in vitro and in vivo. Unpairing in the AT-rich 13mer region is thermodynamically stable even in the absence of DnaA protein, but only when divalent cations are omitted from the reaction. In the presence of Mg2+, oriC melting is strictly DnaA dependent. The sensitive region is distinct from that detected in the absence of DnaA as it is located further to the left within the minimal origin. In addition, the DNA is severely distorted between the three 13mers and the IHF binding site in oriC. A change of conformation can also be observed during the initiation of DNA replication in vivo. This is the first in vivo evidence for a structural change at the 13mers during initiation complex formation.     

Stoichiometry of DnaA and DnaB protein in initiation at the Escherichia coli chromosomal origin

Initiation of DNA replication at the Escherichia coli chromosomal origin, oriC, occurs through an ordered series of events that depend first on the binding of DnaA protein, the replication initiator, to DnaA box sequences within oriC followed by unwinding of an AT-rich region near the left border. The prepriming complex then forms, involving the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork. We assembled and isolated the prepriming complexes on an oriC plasmid, then determined the stoichiometries of proteins in these complexes by quantitative immunoblot analysis. DnaA protein alone binds to oriC with a stoichiometry of 4-5 monomers per oriC DNA. In the prepriming complex, the stoichiometries are 10 DnaA monomers and 2 DnaB hexamers per oriC plasmid. That only two DnaB hexamers are bound, one for each replication fork, suggests that the binding of additional molecules of DnaA in forming the prepriming complex restricts the loading of additional DnaB hexamers that can bind at oriC.     

Mutations predetermined by the primary structure of DNA

This study is concerned with an experimental verification of hypotheses postulating the involvement of self-complementary nucleotide sequences in the formation of deletions and insertions. It was suggested that deletions can arise in the regions of self-complementary nucleotide sequences, which allows the formation of the hairpin structures in a single-stranded DNA, arising during excision repair. These hairpin structures can be eliminated by nucleases or during DNA replication. Insertions can arise as a result of homologous recombination, when a migrating DNA strand contains a self-complementary sequence which forms hairpin structure. Model experiments were carried out with the pBR322 plasmid. A plasmid DNA with premutational damage in the palindrome-containing region was constructed by in vitro dimethylsulfate modification of one strand of EcoRI-BamHI restriction fragment. The plasmid was used for transformation of Escherichia coli. Restriction mapping and nucleotide analysis of the mutant DNAs demonstrated that they all contained deletions. The end points of the deletions coincide with the palindrome. To model homologous recombination, a plasmid with D-loop was constructed. A single-stranded DNA fragment containing palindrome forming a hairpin structure was introduced into the plasmid DNA and covalently fixed in the complex. When E. coli cells were transfected with this DNA, plasmid mutants containing insertions predetermined by palindromic structure arose. The evolutionary role of mutations predetermined by primary DNA structure is discussed.     

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.     

Role of single-stranded DNA binding activity of T antigen in simian virus 40 DNA replication

We have previously mapped the single-stranded DNA binding domain of large T antigen to amino acid residues 259 to 627. By using internal deletion mutants, we show that this domain most likely begins after residue 301 and that the region between residues 501 and 550 is not required. To study the function of this binding activity, a series of single-point substitutions were introduced in this domain, and the mutants were tested for their ability to support simian virus 40 (SV40) replication and to bind to single-stranded DNA. Two replication-defective mutants (429DA and 460EA) were grossly impaired in single-stranded DNA binding. These two mutants were further tested for other biochemical activities needed for viral DNA replication. They bound to origin DNA and formed double hexamers in the presence of ATP. Their ability to unwind origin DNA and a helicase substrate was severely reduced, although they still had ATPase activity. These results suggest that the single-stranded DNA binding activity is involved in DNA unwinding. The two mutants were also very defective in structural distortion of origin DNA, making it likely that single-stranded DNA binding is also required for this process. These data show that single-stranded DNA binding is needed for at least two steps during SV40 DNA replication.     

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32

The chromosomal replication origins (oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.     

DNA replication termination in Escherichia coli parB (a dnaG allele), parA, and gyrB mutants affected in DNA distribution.

We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication. In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction. To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine. In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal. In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC. In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes. This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature. Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography. The DNA in the filaments was either centrally located or distributed throughout. The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E. coli primase. It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.     

Specificity of the S1 nuclease from Aspergillus oryzae.

Conditions are described for digesting single-stranded DNA by S1 nuclease without introducing breaks in double-stranded DNA. The enzyme is inhibited by low concentrations of various compounds of phosphate. Under certain conditions S1 nuclease cleaves the strand opposite a nick in bacteriophage T5 DNA; under other conditions, the enzyme cleaves a loop in one strand of heteroduplex lambdaDNA while leaving the opposite strand intact. S1 nuclease makes many single strand breaks in ultraviolet-irradiated duplex lambdaDNA. Superhelical DNA of phiX174 (Form I) is converted first to a relaxed circular molecule (Form II), and then to a linear molecule (Form III) by cleavage at one site per molecule. Since the cleavage occurs at many sites in the population of molecules, the partially single-stranded regions in phiX174 superhelical DNA are not determined by specific nucleotide sequences.     

Assembly of genes from partially overlapping fragments using single-stranded DNA and sequence-specific synthetic oligodeoxynucleotides

A simple procedure for the precise assembly of functional DNA sequences from overlapping fragments is described. The fragments to be joined are cloned in tandem in the proper relative orientation into a vector from which single-stranded DNA copies can be obtained. Single-stranded DNA is cut by a restriction enzyme at corresponding sites in the two overlap regions, which are made double-stranded by annealing an oligonucleotide of appropriate sequence to them. This results in the excision of the unwanted sequences between the two overlap regions. After removal of the restriction enzyme the DNA is reannealed using the same oligonucleotide, ligated to give closed circular molecules and used to transform competent cells. Clones with the desired structure appear in the progeny at high frequency. The method has the advantage that restriction enzymes with short recognition sequences, cutting frequently in the target DNA, can be used and hence the overlap region required can be quite short.     

Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA

Regions of bacterial chromosomes occupy characteristic locations within the cell. In Bacillus subtilis, the origin of replication, oriC, is located at 0 degrees /360 degrees on the circular chromosome. After duplication, sister 0 degrees regions rapidly move to and then reside near the cell quarters. It has been hypothesized that origin function or oriC sequences contribute to positioning and movement of the 0 degrees region. We found that the position of a given chromosomal region does not depend on initiation of replication from the 0 degrees region. In an oriC mutant strain that replicates from a heterologous origin (oriN) at 257 degrees , the position of both the 0 degrees and 257 degrees regions was similar to that in wild-type cells. Thus, positioning of chromosomal regions appears to be independent of which region is replicated first. Furthermore, we found that neither oriC sequences nor the replication initiator DnaA is required or sufficient for positioning a region near the cell quarters. A sequence within oriC previously proposed to play a critical role in chromosome positioning and partitioning was found to make little, if any, contribution. We propose that uncharacterized sites outside of oriC are involved in moving and/or maintaining the 0 degrees region near the cell quarters.     

Effect of terminal non-homology on intramolecular recombination of linear plasmid substrates in Escherichia coli.

Circular dimer plasmids linearized with a restriction endonuclease undergo intramolecular recombination to yield recombinant circular monomers at high efficiency by a recA-independent mechanism in Escherichia coli recB recC sbcA mutants. The rate of this reaction is at least 1000-fold higher than the recombination rate observed for circular plasmid recombination substrates in the same mutants. Three potential models have been previously proposed to explain the recombination events observed. The validity of these models was tested in recA recB recC sbcA mutants using additional recombination substrates. These substrates, when linearized by incubation with an appropriate restriction enzyme, contain non-homologous adenovirus 2 DNA on one or both ends. The data indicate that terminal non-homology does not significantly affect the efficiency of recovering recombinants. In contrast to many recombination models proposed that involve the invasion of homologous duplex DNA by single-stranded DNA ends, the intramolecular recombination reaction studied here does not appear to involve direct pairing from the end(s) of the substrate DNA. Furthermore, the results are consistent with a model proposing that pairing and strand exchange occur between two homologous duplex regions within the linear dimer molecule.     

Analysis of DNA replication forks encountering a pyrimidine dimer in the template to the leading strand.

Electron microscopy (EM) was used to visualize intermediates of in vitro replication of closed circular DNA plasmids. Cell-free extracts were prepared from human cells that are proficient (IDH4, HeLa) or deficient (CTag) in bypass replication of pyrimidine dimers. The DNA substrate was either undamaged or contained a single cis, syn thymine dimer. This lesion was inserted 385 bp downstream from the center of the SV40 origin of replication and sited specifically in the template to the leading strand of the newly synthesized DNA. Products from 30 minute reactions were crosslinked with psoralen and UV, linearized with restriction enzymes and spread for EM visualization. Extended single-stranded DNA regions were detected in damaged molecules replicated by either bypass-proficient or deficient extracts. These regions could be coated with Escherichia coli single-stranded DNA binding protein. The length of duplex DNA from a unique restriction site to the single-stranded DNA region was that predicted from blockage of leading strand synthesis by the site-specific dimer. These results were confirmed by S1nuclease treatment of replication products linearized with single cutting restriction enzymes, followed by detection of the diagnostic fragments by gel electrophoresis. The absence of an extended single-stranded DNA region in replication forks that were clearly beyond the dimer was taken as evidence of bypass replication. These criteria were fulfilled in 17 % of the molecules replicated by the IDH4 extract.     

An essential tryptophan of Escherichia coli DnaA protein functions in oligomerization at the E. coli replication origin

In the initiation of bacterial DNA replication, DnaA protein recruits DnaB helicase to the chromosomal origin, oriC, leading to the assemble of the replication fork machinery at this site. Because a region near the N terminus of DnaA is required for self-oligomerization and the loading of DnaB helicase at oriC, we asked if these functions are separable or interdependent by substituting many conserved amino acids in this region with alanine to identify essential residues. We show that alanine substitutions of leucine 3, phenylalanine 46, and leucine 62 do not affect DnaA function in initiation. In contrast, we find on characterization of a mutant DnaA that tryptophan 6 is essential for DnaA function because its substitution by alanine abrogates self-oligomerization, resulting in the failure to load DnaB at oriC. These results indicate that DnaA bound to oriC forms a specific oligomeric structure, which is required to load DnaB helicase.