RecA-mediated rescue of Escherichia coli strains with replication forks arrested at the terminus

The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1(CW)) or counterclockwise (intR1(CC)) orientations. Only the intR1(CC) strain, with replication forks arrested at the terminus, required RecA for survival. Unlike the strains with RecA-dependent replication known so far, the intR1(CC) strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability. The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC and intR1(CC) strains. In the intR1(CC) strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus. This was consistent with suppression of the RecA dependence of the intR1(CC) strain by inactivating Tus, the protein needed to block replication forks at Ter sites. Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways.     

Proteins encoded by the Escherichia coli replication terminus region.

The replication terminus region (31 to 35 min) of the Escherichia coli chromosome contains very few mapped genes (two per min) compared with the remainder of the chromosome, and much of the DNA appears dispensable. In order to determine whether, despite this, the terminus region consists of protein-coding sequences, we cloned 44 kb (1 min) of terminus region DNA (that surrounding trg at 31.4 min) and examined its ability to catalyze protein synthesis in vitro or in minicells. We were able to account for more than half the coding capacity of the cloned DNA with proteins synthesized in these systems, indicating that the sparsity of mapped genes in the terminus region does not result from a lack of identifiable coding sequences. We can therefore conclude that the terminus region is composed mainly of expressable, albeit inessential, protein-encoding genes.     

Molecular cloning of the region of the terminus of Escherichia coli K-12 DNA replication.

A series of plasmids have been isolated either by ligation of defined restriction fragments to plasmid pBR325 or by screening of a cosmid bank by in situ colony hybridization. Together with one previously isolated plasmid, they spanned 86% of the 30.5- to 34-min region of the genetic map of Escherichia coli K-12. Physical analysis of these plasmids and hybridizations to Southern blots confirmed the endonuclease map of this region, with the exception of a 9.3-kilobase pair inversion.     

Cloning of the contiguous 165-kilobase-pair region around the terminus of Escherichia coli K-12 DNA replication.

Escherichia coli K-12 chromosomal DNA was partially digested with either EcoRI or HindIII, and cosmid libraries were constructed. By screening these libraries, a series of partially overlapping clones which covered the terC region was isolated. The cloned area spanned about 165 kilobase pairs, corresponding to the 29.7-to-33.2-min region of the genetic map of the E. coli chromosome.     

Hemimethylation prevents DNA replication in E. coli

The DNA adenine methylase of E. coli methylates adenines at GATC sequences. Strains deficient in this methylase are transformed poorly by methylated plasmids that depend on either the pBR322 or the chromosomal origins for replication. We show here that hemimethylated plasmids also transform dam- bacteria poorly but that unmethylated plasmids transform them at high frequencies. Hemimethylated daughter molecules accumulate after the transformation of dam- strains by fully methylated plasmids, suggesting that hemimethylation prevents DNA replication. We also show that plasmids purified from dam+ bacteria are hemimethylated at certain sites. These results can explain why newly formed daughter molecules are not substrates for an immediate reinitiation of DNA replication in wild-type E. coli.     

Isolation of DNA fragments containing replicating growing forks from both E. coli and B. subtilis

Summary By the use of a restriction enzyme digestion of gently lysed E. coli or B. subtilis cells, it is possible to isolate a minute fraction of the total DNA that has an unusually high sedimentation coefficient. Upon inspection of this DNA in the electron microscope, branched DNA fragments are observed. Single branched DNA fragments were analyzed by restriction enzyme and partial denaturation mapping techniques. The fragments appear to have the properties of growing forks excised from in vivo replicating intermediates. In B. subtilis, the minute fraction of DNA was also investigated by transformation assays and found to be greatly enriched for a marker near the origin and slightly enriched for a terminus marker.     

The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate

A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks. Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone. Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E. coli proteins (replication factor Y (protein n'), proteins n and n', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein. Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules. The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides. The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.     

DNA sequence of the 5'' terminus containing the replication origin of parvovirus replicative form DNA.

The nucleotide sequence of the 5' terminus of the parvovirus H-1 was determined. There are two orientations of the 242-base-pair terminal palindrome in native replicative form DNA, one inverted with respect to the other. Adjacent to the terminal palindrome is an AT-rich region that is noncoding and contains a 55-base-pair tandem repeat. The addition mutant of H-1, DI-1, was also sequenced in this region and shown to have three copies of the tandem repeat sequence. Similarly, the related parvovirus H-3 contains only one copy of this repeat sequence. This region contains the replication origin for parvovirus replicative form DNA replication. Some of the implications of these results are discussed.     

dnaC-dependent reconstitution of replication forks in Escherichia coli lysates.

Lysates of Escherichia coli exhibit a DNA-synthesizing activity that depends on the presence of replication forks and of replication proteins. Replicative activity was reconstituted in vitro by mixing lysates prepared from temperature-sensitive dnaB mutants with wild-type dnaB protein. Lysates of double mutants deficient in both dnaB and dnaC genes could only be complemented by the addition of both dnaB and dnaC proteins, whereas lysates deficient in dnaC protein did not require the addition of any exogenous factor. This shows that the replication machinery, once it is running along the chromosome, is independent of dnaC protein, dnaC activity, however, is required for the replacement of defective dnaB protein at running replication forks.     

A critical role of the 3' terminus of nascent DNA chains in recognition of stalled replication forks

Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3' termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative "3' terminus binding pocket" present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses.     

The 245 base-pair oriC sequence of the E. coli chromosome directs bidirectional replication at an adjacent region

The replication origin of the E. coli K-12 chromosome has been isolated as autonomously replicating molecules(oriC plasmid), and the DNA region essential for replicating function(oriC) has been localized to a sequence of 232-245 base-pairs(bp) by deletion analysis. In this report, the functional role of oriC was analysed by using an in vitro replication system and various OriC+ and OriC- plasmids previously constructed. The results obtained were summarized as follows: (1) The oriC sequence contained information enough to direct bidirectional replication. (2) The actual DNA replication began at a region near, but outside, oriC and progressed bidirectionally. (3) Initiation of DNA synthesis at the specific region required the dnaA-complementing fraction from cells harboring a dnaA-carrying plasmid.     

Thylakoid-bound chloroplast DNA from spinach is enriched for replication forks

Chloroplast DNA is bound to the thylakoids of spinach chloroplasts. To examine a possible role for thylakoid-bound DNA in chloroplast DNA replication, vesicles formed by treating chloroplasts in 3.5 mM MgCl2 were used. Chloroplast DNA fragments are bound to the surface of these vesicles. Chloroplast DNA isolated from vesicles that had been first treated with Eco R1 contained 10% of branched fragments whereas chloroplast DNA isolated from intact chloroplasts and treated with Eco R1 contained 2% of branched fragments. This result is consistent with the growing replication fork of chloroplast DNA being associated with the chloroplast internal membrane system. Branched fragments from the chloroplast DNA digested with Eco R1 prior to the isolation from the vesicle contained fragments of unequal length. Membrane binding in chloroplasts may have a similar role in DNA replication as it does in bacteria.     

Site-specific recombination in the replication terminus region of Escherichia coli: functional replacement of dif.

The replication terminus region of the Escherichia coli chromosome encodes a locus, dif, that is required for normal chromosome segregation at cell division. dif is a substrate for site-specific recombination catalysed by the related chromosomally encoded recombinases XerC and XerD. It has been proposed that this recombination converts chromosome multimers formed by homologous recombination back to monomers in order that they can be segregated prior to cell division. Strains mutant in dif, xerC or xerD share a characteristic phenotype, containing a variable fraction of filamentous cells with aberrantly positioned and sized nucleoids. We show that the only DNA sequences required for wild-type dif function in the terminus region of the chromosome are contained within 33 bp known to bind XerC and XerD and that putative active site residues of the Xer recombinases are required for normal chromosome segregation. We have also shown that recombination by the loxP/Cre system of bacteriophage P1 will suppress the phenotype of a dif deletion strain when loxP is inserted in the terminus region. Suppression of the dif deletion phenotype did not occur when either dif/Xer or loxP/Cre recombination acted at other positions in the chromosome close to oriC or within lacZ, indicating that site-specific recombination must occur within the replication terminus region in order to allow normal chromosome segregation.     

Nucleotide sequence of the replication region from the Mycobacterium-Escherichia coli shuttle vector pEP2

The replication region derived from the Corynebacterium diphtheriae-Escherichia coli plasmid pNG2 was sequenced. This 1.85-kb segment contains a single origin of DNA replication, one major open reading frame and shares no sequence homology with previously described plasmids.     

DNA replication: making two forks from one prereplication complex

The copying of chromosomal DNA initiates from a single nucleoprotein assembly called the prereplication complex. New findings in a recent issue of Molecular Cell (Yardimci et?al., 2010) reveal that this complex dissolves into two independent replisomes that move away from each other as DNA synthesis ensues.     

Expression of the replication region of phage lambda DNA cloned into pBR322 in E. coli minicells

Replication region of bacteriophage lambda DNA was cloned into pBR322 plasmid by the use of two restriction enzymes--PstI and HindIII. The restriction analysis of four obtained plasmids revealed that lambda DNA was cloned in both orientations. Recombinant plasmids were transferred to the minicell-producing strain of E. coli and synthesis of the plasmid-mediated proteins was analysed by polyacrylamide-gel electrophoresis. All four recombinant plasmids produced lambda DNA replication proteins pO and pP as well as some proteins specific for pBR322. The orientation of cloned fragment did not affect the synthesis of lambda DNA replication proteins.