Plasmid replication-associated single-strand-specific methyltransferases

Abstract Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M.BceJIII and M.EcoGIX, using artificial constructs. Mating properties of engineered pESBL derivatives were also investigated. Both MTases yield promiscuous m6A modification of single strands, in the context SAY (where S = C or G and Y = C or T). Strikingly, this methylation is asymmetric in vivo, detected almost exclusively on one DNA strand, and is incomplete: typically, around 40% of susceptible motifs are modified. Genetic and biochemical studies suggest that enzyme action depends on replication mode: DNA Polymerase I (PolI)-dependent ColE1 and p15A origins support asymmetric modification, while the PolI-independent pSC101 origin does not. An MTase-PolI complex may enable discrimination of PolI-dependent and independent plasmid origins. M.EcoGIX helps to establish pESBL in new hosts by blocking the action of restriction enzymes, in an orientation-dependent fashion. Expression and action appear to occur on the entering single strand in the recipient, early in conjugal transfer, until lagging-strand replication creates the double-stranded form.

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Exonuclease III (XthA) EnforcesIn VivoDNA Cloning ofEscherichia coliTo Create Cohesive Ends

Journal of Bacteriology ◽

10.1128/jb.00660-18 ◽

2018 ◽

Vol 201 (5) ◽

Cited By ~ 9

Author(s):

Shingo Nozaki ◽

Hironori Niki

Keyword(s):

Dna Polymerase ◽

Dna Fragments ◽

Dna Polymerase I ◽

Dna Cloning ◽

Content Type ◽

E Coli ◽

Polymerase I ◽

Cloning Technology

ABSTRACTEscherichia colihas an ability to assemble DNA fragments with homologous overlapping sequences of 15 to 40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA cloning technology. However, the molecular mechanism by whichE. coliaccomplishes such cloning is still unknown. In this study, we provide evidence that thein vivocloning ofE. coliis independent of both RecA and RecET recombinases but is dependent on XthA, a 3′ to 5′ exonuclease. Here,in vivocloning ofE. coliby XthA is referred to asin vivoE. colicloning (iVEC). We also show that iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced intoE. colicells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development ofin vivoDNA cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.IMPORTANCECloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently, anin vitrorecombination system for DNA cloning was shown to enable the joining of multiple DNA fragments at once. Interestingly,E. colipotentially assembles multiple linear DNA fragments that are introduced into the cell. Improved protocols for thisin vivocloning have realized a high level of usability, comparable to that byin vitrorecombination reactions. However, the mechanism ofin vivocloning is highly controversial. Here, we clarified the fundamental mechanism underlyingin vivocloning and also constructed a strain that was optimized forin vivocloning. Additionally, we streamlined the procedure ofin vivocloning by using a single microcentrifuge tube.

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Exonuclease III (XthA) enforcesin vivoDNA cloning ofEscherichia colito create cohesive ends

10.1101/454074 ◽

2018 ◽

Author(s):

Shingo Nozaki ◽

Hironori Niki

Keyword(s):

Dna Polymerase ◽

Recombination Reaction ◽

Dna Fragments ◽

Dna Polymerase I ◽

Dna Cloning ◽

E Coli ◽

Polymerase I ◽

Cloning Technology

AbstractEscherichia colihas an ability to assemble DNA fragments with homologous overlapping sequences of 15-40 bp at each end. Several modified protocols have already been reported to improve this simple and useful DNA-cloning technology. However, the molecular mechanism by whichE. coliaccomplishes such cloning is still unknown. In this study, we provide evidence that thein vivocloning ofE. coliis independent of both RecA and RecET recombinase, but is dependent on XthA, a 3’ to 5’ exonuclease. Here, in vivocloning ofE. coliby XthA is referred to as iVEC (in vivo E. colicloning). Next, we show that the iVEC activity is reduced by deletion of the C-terminal domain of DNA polymerase I (PolA). Collectively, these results suggest the following mechanism of iVEC. First, XthA resects the 3′ ends of linear DNA fragments that are introduced intoE. colicells, resulting in exposure of the single-stranded 5′ overhangs. Then, the complementary single-stranded DNA ends hybridize each other, and gaps are filled by DNA polymerase I. Elucidation of the iVEC mechanism at the molecular level would further advance the development ofin vivoDNA-cloning technology. Already we have successfully demonstrated multiple-fragment assembly of up to seven fragments in combination with an effortless transformation procedure using a modified host strain for iVEC.ImportanceCloning of a DNA fragment into a vector is one of the fundamental techniques in recombinant DNA technology. Recently,in vitrorecombination of DNA fragments effectively joins multiple DNA fragments in place of the canonical method. Interestingly,E. colican take up linear double-stranded vectors, insert DNA fragments and assemble themin vivo.Thein vivocloning have realized a high level of usability comparable to that byin vitrorecombination reaction, since now it is only necessary to introduce PCR products intoE. colifor thein vivocloning. However, the mechanism ofin vivocloning is highly controversial. Here we clarified the fundamental mechanism underlyingin vivocloning of E. coli and also constructed anE. colistrain that was optimized forin vivocloning.

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Site-specific modification of E. coli DNA polymerase I large fragment with pyridoxal 5'-phosphate

Biochemistry ◽

10.1021/bi00304a030 ◽

1984 ◽

Vol 23 (9) ◽

pp. 2073-2078 ◽

Cited By ~ 14

Author(s):

Anup K. Hazra ◽

Sevilla Detera-Wadleigh ◽

Samuel H. Wilson

Keyword(s):

Dna Polymerase ◽

Large Fragment ◽

Dna Polymerase I ◽

Site Specific ◽

E Coli ◽

Specific Modification ◽

Polymerase I

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Genetic evidence for two protein domains and a potential new activity in bacteriophage T4 DNA polymerase.

Genetics ◽

10.1093/genetics/124.2.213 ◽

1990 ◽

Vol 124 (2) ◽

pp. 213-220 ◽

Cited By ~ 2

Author(s):

L J Reha-Krantz

Keyword(s):

Dna Polymerase ◽

Bacteriophage T4 ◽

Rnase H ◽

Ribonuclease H ◽

Temperature Sensitive ◽

Polymerase Gene ◽

Dna Polymerase I ◽

E Coli ◽

Intragenic Complementation ◽

Polymerase I

Abstract Intragenic complementation was detected within the bacteriophage T4 DNA polymerase gene. Complementation was observed between specific amino (N)-terminal, temperature-sensitive (ts) mutator mutants and more carboxy (C)-terminal mutants lacking DNA polymerase polymerizing functions. Protein sequences surrounding N-terminal mutation sites are similar to sequences found in Escherichia coli ribonuclease H (RNase H) and in the 5'----3' exonuclease domain of E. coli DNA polymerase I. These observations suggest that T4 DNA polymerase, like E. coli DNA polymerase I, contains a discrete N-terminal domain.

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