A DNA primase specified by I-like plasmids.

Lanka, Erich; Scherzinger, Eberhard; Günther, E; Schuster, Hans‐Uwe

doi:10.1073/pnas.76.8.3632

Cited by 42 publications

(19 citation statements)

References 31 publications

(15 reference statements)

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“…Because of the biological parallel between F and the I-like conjugative plasmids, it also seems possible that the Tral protein has DNA primase activity. It has been concluded that F does not encode a DNA primase, since it did not suppress a chromosomal dnaG temperature-sensitive mutation (36), nor did extracts from cells containing the F-like plasmids Rldrd-16 or R100-1 provide DNA priming activity in an in vitro DNA replication system (21). However, the plasmid specificity of traI might have defeated either of these attempts to detect an F-like-plasmid-encoded DNA primase.…”

Section: Resultsmentioning

confidence: 99%

Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport

Traxler

Minkley

1987

J Bacteriol

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The DNA transfer stage of conjugation requires the products of the F sex factor genes traMYDIZ and the cis-acting site oriT. Previous interpretation of genetic and protein analyses suggested that traD, tral, and traZ mapped as contiguous genes at the distal end of the transfer operon and saturated this portion of the F transfer region (which ends with an IS3 element). Using antibodies prepared against the purified TraD and TraI proteins, we analyzed the products encoded by a collection of chimeric plasmids constructed with various segments of traDIZ DNA. We found the tral gene to be located 1 kilobase to the right of the position suggested on previous maps. This creates an unsaturated space between traD and tral where unidentified Ira genes may be located and leaves insufficient space between tral and 1S3 for coding the 94-kilodalton protein previously thought to be the product of traZ. We found that the 94-kilodalton protein arose from a translational restart and corresponds to the carboxy terminus of tral; we named it TraI*. The precise physical location of the traZ gene and the identity of its product are unknown. The onT nicking activity known as TraZ may stem from unassigned regions between traD and tral and between tral and IS3, but a more interesting possibility is that it is actually a function of tral. On our revised map, the position of a previously detected RNA polymerasebinding site corresponds to a site at the amino terminus of Iral rather than a location 1 kilobase into the coding region of the gene. Furthermore, the physical and genetic comparison of the F traD and tral genes with those of the closely related F-like conjugative plasmids Rl and R100 is greatly simplified. The translational organization we found for tral, together with its identity as the structural gene for DNA helicase I, suggests a possible functional link to several other genes from which translational restart polypeptides are expressed.These include the primases of the conjugative plasmids Coil and R16, the primase-helicase of bacteriophage T7, and the cisA product (nickase) of phage +X174.

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Section: Resultsmentioning

confidence: 99%

Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport

Traxler

Minkley

1987

J Bacteriol

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“…Hence, the primase protein is transferred in noncatalytic amounts, and in addition, at least an equal number of Sog-160 molecules is transferred. It is known that Incll plasmid primase protein purifies as a DNA-binding protein (21). Therefore a substantial fraction of the transferred single-stranded DNA may be complexed with Sog proteins.…”

Section: Discussionmentioning

confidence: 99%

Transfer of tra proteins into the recipient cell during bacterial conjugation mediated by plasmid ColIb-P9

Rees

Wilkins

1989

J Bacteriol

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Selective transfer of the two products of the ColIb primase gene, sog, from donor to recipient cell during conjugation was demonstrated by two independent methods. The transfer of these tra proteins was unidirectional and dependent on DNA transfer. The Sog polypeptides were localized to the cytoplasm of the donor cell, but they appeared to interact with other tra gene products located in the inner membrane. After cell mating, the transferred polypeptides were found to be in the cytoplasm of the recipient cell, and it is estimated that as many as 500 Sog polypeptides were transferred per round of conjugation. It is proposed that these proteins are transferred as a result of an interaction with the single-stranded DNA and that the transferred strand may be coated with Sog polypeptides.During bacterial conjugation mediated by Incll plasmids such as ColIb-P9, a specific plasmid strand is transferred to the recipient cell, where the double-stranded DNA molecule is regenerated (31). DNA synthesis on the transferred strand is initiated by a plasmid-encoded DNA primase that is the product of a gene called sog (11). This gene is a component of the I1 conjugation (tra) system, and it specifies two large, in-phase translation products of Mr 210,000 and 160,000, which are designated here 23,27,32). The primase generates short RNA primers, and this activity is dependent on the N-terminal third of 20,32).To initiate DNA synthesis on the transferred plasmid strand, sog DNA primase is supplied by the donor cell and transmitted to the recipient bacterium. The primase is remarkable in this respect because no general transfer of proteins occurs during bacterial conjugation (26,28,29). Primase transfer was first inferred from a functional test which showed that the enzyme originating from the donor strain could restore bacterial DNA synthesis in mated recipients defective in Escherichia coli primase (12). Subsequently, it was demonstrated that Sog-210 and Sog-160 from labeled donor cells were present in a set of proteins selectively retained by recipients after conjugation (23).The objective of this work was to characterize further the transfer of Sog polypeptides during bacterial conjugation. Identification of transferred proteins relies on separation of donor and recipient after mating. This was achieved either by destroying the labeled donor cells by lysis-from-without with bacteriophage T6 or by using minicells as the recipient cells and separating the cells according to size. This second method exploits the finding that minicells are competent recipients in matings mediated by Incll plasmids (15). We have investigated the location of the Sog polypeptides both in the donor cell and in the recipient cell after transfer. We have also determined their direction of transfer relative to that of the DNA and the number of Sog polypeptide molecules transmitted per conjugation cycle. Media, enzymes, and radiochemicals. Media and general culture conditions were as described previously (11,23). SGC is an M9 salts-glucose (0.4%)-Casamino A...

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“…In combination with the protein concentration of the extracts measured by the method of Lowry et al (32), these values served to calculate the gp␣ concentrations. DNA primase was assayed in vitro in the presence of rifampin in an extract of E. coli dnaB dnaC mutant strain BC1304 as described previously (28,29). Viral fd DNA was used as the template for the primase and complementary-strand synthesis.…”

Section: Resultsmentioning

confidence: 99%

Domain structure of phage P4 alpha protein deduced by mutational analysis

et al. 1995

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Bacteriophage P4 DNA replication depends on the product of the ␣ gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the ␣ gene. Sequence analysis of the entire gene predicted a domain structure for the ␣ polypeptide chain (777 amino acid residues, M r 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the ␣ polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for ␣ mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.Bacteriophage P4 is a temperate satellite phage of Escherichia coli that relies on morphogenetic and lysis functions of the helper phage P2 for its propagation (30). The P4 prophage state is maintained either by integration into the host chromosome or by autonomous replication as a multicopy plasmid. Lysogenization of the host, DNA replication during the lytic cycle, and establishment of the plasmid state all occur independently of helper phage (30). Replication of P4 DNA requires only the P4 ␣ gene product (gp␣) and two cis-acting elements, namely, the origin of replication ori and the sequence-related region crr (cis replication region) (7,26,30,63). Another P4 gene, cnr (copy number regulation), maps directly upstream of ␣; its product has been suggested to control P4 DNA replication, in particular at the plasmid level, maybe by direct interaction of Cnr with gp␣ (59). Thus, protein-protein interaction might modulate at least one specific function of the ␣ protein.The ␣ protein is a multifunctional phage replication protein that recognizes ori and crr by binding to octamer sequences (type I repeats, TGTTCACC [16]) (64). In addition, gp␣ catalyzes the unwinding of DNA via its helicase activity. DNA is unwound with a 3Ј to 5Ј polarity with respect to the strand that gp␣ has bound. The helicase action is fueled by nucleoside triphosphate (NTP) hydrolysis of an interrelated singlestranded DNA-dependent NTPase. gp␣ also functions as a primase, with a template-dependent oligonucleotide-synthesizing activity (56, 64). The primase activity of gp␣ can be replaced by the primase of the host, the DnaG protein (56). However, the P4 burst sizes in the presence of a primase-null ␣ protein and an active DnaG protein were always small, indicating that DnaG can only partially substitute for the primase fu...

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A DNA primase specified by I-like plasmids.

Cited by 42 publications

References 31 publications

Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport

Revised genetic map of the distal end of the F transfer operon: implications for DNA helicase I, nicking at oriT, and conjugal DNA transport

Transfer of tra proteins into the recipient cell during bacterial conjugation mediated by plasmid ColIb-P9

Domain structure of phage P4 alpha protein deduced by mutational analysis

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