The bond in the bacteriophage øX174 gene A protein‐DNA complex is a tyrosyl‐5'‐phosphate ester

Mansfeld, A.D.M. van; Teeffelen, H.A.A.M. van; Baas, Pieter; Veeneman, G. H.; Boom, Jacques H. van; Jansz, H.S.

doi:10.1016/0014-5793(84)80804-2

Cited by 27 publications

(10 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions [22][23][24][25][26][27][28][29][30] in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.…”

mentioning

confidence: 99%

Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site‐specific DNA‐cleaving/DNA‐joining activity

Scherzinger

Kruft

Otto

1993

European Journal of Biochemistry

View full text Add to dashboard Cite

A site-specific and strand-specific nick, introduced into the RSFlOlO plasmid origin of transfer ( o r i v , initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this niclung at the duplex oriT in vitro using purified preparations of the three known RSF1010-mobilization proteins: MobA (78-kDa form of RSFlOlO primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrinski, B. (1992) Nucleic Acids Res. 20,. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78-kDa protein by itself is capable of creating the oriT-specific nick if the DNA is present in the singlestranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5' or 3' end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11 -residue oligonucleotide AAGTGCGC-CCT which is cleaved at the 3' side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5'-phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3' end at least the seven nucleotides upstream of the nick site. The covalent MobA-DNA linkage has been determined by two-dimensional thin-layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the 32P-labeled MobA-oligonucleotide complex with lysine carboxypeptidase yielded a single DNA-bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22-30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.RSFlOlO is a broad-host-range IncQ plasmid (nearly identical to R1162) that is efficiently mobilized during the conjugal transfer of IncP-1 group plasmids. The mobilization of RSFlOlO requires a 38-bp site, oriT on the plasmid as well as the products of the RSFlOlO genes mobA, mobB, and mobC [l, 21. During mobilization, oriT is nicked at the position indicated in Fig. 3 (the nic site) [2, 31. The interrupted DNA strand is subsequently exported, with its 5' end first, to a recipient cell and, after recircularization, converted by DNA synthesis to a double strand [4].The enzyme responsible for generation of the orirspecific nick is MobA. In vitro, superhelical or linear RSFlOlO DNA and purified MobA, MobB and MobC proteins form a cleavable complex in the presence of Mg", which is characterized by its sensitivity to protein-denaturant treatment. On addition of alkali or detergent to such a complex, a singlestrand break is generated in the DNA at the site correspond-

show abstract

mentioning

confidence: 99%

Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site‐specific DNA‐cleaving/DNA‐joining activity

Scherzinger

Kruft

Otto

1993

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…This octadecamer corresponds to the first 18 nucleotides of the ¢X174 replication origin. Upon incubation with gene A protein or A* protein this oligonucleotide is specifically cleaved after the G residue [25]. After cleavage gene A protein and A* protein are radioactive due to covalent linkage of the radioactive oligonucleotide [32p]ATATTAATAAC to a tyrosyl residue of these proteins.…”

Section: Resultsmentioning

confidence: 99%

Alteration of the ATG start codon of the A* protein of bacteriophage ϕX174 into an ATT codon yields a viable phage indicating that A* protein is not essential for ϕX174 reproduction

et al. 1987

Self Cite

View full text Add to dashboard Cite

Bacteriophage ϕX174 gene A encodes two proteins: the gene A protein and the smaller A* protein, which is synthesized from a translational start signal within the A gene in the same reading frame as the gene A protein. The gene A protein is involved in initiation, elongation and termination of rolling circle DNA replication. The role of the A* protein in the life cycle of ϕX174, however, is unknown. Using oligonucleotide‐directed mutagenesis a viable ϕXl74 mutant was constructed in which the ATG start codon of the A* protein was changed into an ATT codon. This mutant, ϕX‐4499T, does not synthesize A* protein. The burst size of ϕX‐4499T amounted to 50% of that of wild type ϕXI74. This indicates that A* protein, although advantageous for phage reproduction, is not essential during the life cycle of bacteriophage ϕX174.

show abstract

“…RCR was discovered in ssDNA coliphage ΦX174 some 45 years ago (4)(5)(6). The pioneer characterization of gene A protein made the initiator of ΦX174 RCR the first member of the HUH endonuclease superfamily (7)(8)(9)(10).…”

Section: General Aspects Of Plasmid Rolling-circle Replicationmentioning

confidence: 99%

Plasmid Rolling-Circle Replication

et al. 2015

View full text Add to dashboard Cite

Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

show abstract

The bond in the bacteriophage øX174 gene A protein‐DNA complex is a tyrosyl‐5'‐phosphate ester

Cited by 27 publications

References 25 publications

Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site‐specific DNA‐cleaving/DNA‐joining activity

Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site‐specific DNA‐cleaving/DNA‐joining activity

Alteration of the ATG start codon of the A* protein of bacteriophage ϕX174 into an ATT codon yields a viable phage indicating that A* protein is not essential for ϕX174 reproduction

Plasmid Rolling-Circle Replication

Contact Info

Product

Resources

About