Biomedical subjects

A Abeles

Publications and source records attributed to A Abeles.

8 recordsLinked to original sources

PubMed1995

A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural. DNA preparations relative to controls.

Adenine↗

PubMed1993

Evidence of two levels of control of P1 oriR and host oriC replication origins by DNA adenine methylation.

A mutant mini-P1 plasmid with increased copy number can be established in Dam- strains of Escherichia coli, where mini-P1 plasmid replication is normally blocked. Comparison of this plasmid and a plasmid driven by the host oriC replication origin showed that both origins are subject to control by methylation at two different levels. First, both origins appear to be subject to negative regulation acting at the level of hemimethylation. This probably involves the sequestration of the hemimethylated DNA produced by replication, as has been previously described for oriC. Second, both origins show a positive requirement for adenine methylation for efficient function in vivo. This conclusion is supported by the behavior of the P1 origin in an improved in vitro replication system. In vitro, where sequestration of hemimethylated DNA is not expected to occur, the hemimethylated P1 origin DNA was fully functional as a template. However, the activity of fully unmethylated DNA was severely restricted in comparison with that of either of the methylated forms. This in vitro uncoupling of the two effects of origin methylation suggests that two separate mechanisms are involved.

Adenine↗

PubMed1991

Critical sequences in the core of the P1 plasmid replication origin.

The core of the P1 plasmid replication origin consists of a series of 7-bp repeats and a G+C-rich stretch. Methylation of the GATC sequences in the repeats is essential. Forty different single-base mutations in the region were isolated and assayed for origin function. A single-base change within any 7-bp repeat could block the origin, irrespective of whether GATC bases were affected. The repeats themselves were critical, but the short intervals between them were not. Mutations in the G+C-rich region showed it to be a spacer whose exact length is important but whose sequence can vary considerably. It maintains a precise distance between the 7-bp repeats and binding sites for the P1 RepA initiator protein. It may also serve as a clamp to limit strand separation during initiation.

Bacterial Proteins↗

PubMed1985

The partition functions of P1, P7, and F miniplasmids.

The partition regions of P1, P7, and F miniplasmids are discrete DNA sequences of about 3 kb in length that will promote accurate partition of hybrid plasmids independent of the source of replication functions or the position or orientation of the elements. Each of the par regions seems to be very similarly organized, with open reading frames for essential proteins and a terminal site which appears to be analogous to the centromere of eukaryotic cells. When cloned, these terminal sites exert incompatibility against their respective parent plasmids presumably because they can compete with the parent plasmids as substrates for partition. We have determined the complete DNA sequence of the P1 par region. In addition to the open reading frame for the essential parA protein (42-44 kd), the region contains a second open reading frame which could encode a 38-kd protein. The 2 large open reading frames appear to form an operon that is negatively regulated from a site adjacent to the promoter and responds to the par gene products in trans. Both this site and the downstream "centromere" site, incB, contain blocks of extremely AT-rich sequences, which are postulated to be binding sites for par proteins. The incB and upstream AT-rich regions both contain 20-bp imperfect inverted repeats. Further downstream from the minimal incB sequence (172 bp) lies an additional region which is essential for partition. The further analysis of the P1 par region should be greatly facilitated by the finding that it can function in cis to stabilize pBR322 vectors under conditions where the copy number of pBR322 is reduced.

Amino Acid Sequence↗

PubMed1984

Plasmid P1 replication: negative control by repeated DNA sequences.

The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pieces of the incA fragment that each contain only three repeats destabilize P1 plasmids efficiently. This result makes it unlikely that incA specifies a regulatory product. Our in vivo results suggest that the repeating DNA sequence itself negatively controls replication by titrating a P1-determined protein, RepA, that is essential for replication. Consistent with this hypothesis is the observation that the RepA protein binds to the incA fragment in vitro.

Bacterial Proteins↗

PubMed1983

Partition of unit-copy miniplasmids to daughter cells. I. P1 and F miniplasmids contain discrete, interchangeable sequences sufficient to promote equipartition.

Hybrids formed by insertion of the plasmid maintenance regions of P1 or F into a lambda delta att vector form stable unit-copy plasmids in their Escherichia coli host. They must therefore both be substrates for an accurate cellular partition apparatus that ensures that all daughter cells inherit a plasmid copy. Analysis of deletion mutants of both types of hybrid showed that, although the P1 and F plasmid maintenance regions differ in sequence and specificity, they are similar in general organization. Each contains an approximately 3 X 10(3) base-pair region that is essential for replication (rep) and an adjacent but separable 3 X 10(3) base-pair region that is essential for the stability of plasmid maintenance (par). Each par region is thought to specify the recognition of the plasmid as a substrate for equipartition. The deletion mutants provide sources of isolated rep and par sequences from both P1 and F DNA. These elements were then used to construct composite plasmids with novel combinations and arrangements of rep and par sequences. Heterologous constructions containing P1 rep and F par or F rep and P1 par sequences were maintained faithfully. We conclude that par regions are both necessary and sufficient to promote equipartition of replicating plasmid DNA. This activity is exerted only in cis but otherwise seems to be independent of the position or orientation of the par sequences within the DNA. Both P1 and F par regions include DNA sequences (incB of P1, incD of F) that we propose are analogues of the centromeres of eukaryotic chromosomes. The remaining portions of the par regions are known to encode protein products that, we believe, act at the inc sites. Extra copies of these inc sites appear to exert incompatibility by competition for the cellular partition apparatus.

Bacteriophage lambda↗

PubMed1983

Partition of unit-copy miniplasmids to daughter cells. II. The partition region of miniplasmid P1 encodes an essential protein and a centromere-like site at which it acts.

The stable maintenance of the unit-copy lambda-P1:5R miniplasmid is dependent on adjacent but separable replication (rep) and partition (par) regions of DNA derived from its P1 plasmid parent. The par region consists of an approximately 2.5 X 10(3) base-pair (kb) segment of DNA of which the terminal kb contains the plasmid incompatibility determinant incB. Two of the 14 lambda-P1:5R partition-defective point mutants isolated are amber (nonsense) mutants, showing that a plasmid-encoded protein is essential for proper partition. All of the Par- point mutants are complemented by the wild-type par region in trans. The complementing activity was shown to be an Mr 44,000 protein encoded by the end of the par region distal to incB. Deletion analysis showed that the incB sequence is essential in cis to the plasmid in order that the plasmid be receptive to the par protein. Thus incB appears to be the target site for par protein activity. We propose that the protein binds to incB, forming a complex that is recognized as a substrate for the cellular partition apparatus. The ability of a cloned incB sequence to compete for the par protein or for the cellular partition apparatus accounts for its activity as an incompatibility determinant. The existence of a plasmid-encoded par protein suggests a specific model for equipartition.

Bacteriophage lambda↗

PubMed1982

Genetic and physical map of a P1 miniplasmid.

The prophage form of bacteriophage P1 is a unit-copy plasmid which is maintained with great fidelity in its Escherichia coli host. The plasmid maintenance functions of P1 are clustered in one region of the genome. An 11.5-kilobase fragment from this region has been cloned into a lambda delta att vector and promotes stable unit-copy plasmid maintenance. The properties of the lambda vector facilitated the isolation of deletion mutants affecting the P1 DNA. Twenty-eight deletion mutants were isolated, and their lesions were mapped by physical techniques. The genetic properties of the mutants with respect to plasmid replication, stability of plasmid maintenance, and ability to exert incompatibility effects against P1 and P7 plasmids were determined. These properties, along with those of several subfragments of the P1 insert cloned into high-copy-number plasmid vectors, allow the construction of an unambiguous genetic and physical map of the maintenance functions. A region of less than 3 kilobases, the rep region, is essential for plasmid replication and contains the incA incompatibility determinant within an 800-base-pair segment. Immediately adjacent to rep is a second region of approximately 3 kilobases which is required for stable plasmid maintenance, but not replication. This region, par, contains a second incompatibility element incB which is approximately 1 kilobase in size. The par region appears to specify equipartition of plasmid copies to daughter cells during cell division.

Base Sequence↗