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Identification of a gene encoding the replication initiator protein of the Streptomyces integrating element, pSAM2
J Hagège1, F Boccard, T Smokvina
1Laboratoire de Biologie et Génétique Moléculaire, URA CNRS 1354, Université Paris-Sud, Orsay, France.
Abstract:
pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens which was previously shown to generate single-stranded DNA during replication, indicating that it probably replicates by a rolling-circle replication (RCR) mechanism. Two separate regions are involved in its replication, one of which was shown to contain the plus origin of replication (ds origin). We report here the study of the second region. Its nucleotide sequence was determined and analysed for open reading frames (ORFs). Three putative ORFs were identified: orf183 (183 amino acids (aa)), orf50 (50 aa), and repSA (459 aa). orf183 is not necessary for replication. The function of orf50 is unknown. repSA is essential for pSAM2 replication; it could encode a protein, RepSA, presenting similarities to the replication initiator proteins (Rep) of elements that replicate by an RCR mechanism. A derivative consisting of repSA, the region containing ds origin, a Streptomyces antibiotic resistance marker, and pBR322, could replicate in Streptomyces, further demonstrating that this ORF encodes the major replication protein of pSAM2. repSA might be co-transcribed with the genes involved in integration and excision of pSAM2.
Insights
The pSAM2 integrating element uses rolling-circle replication (RCR). A newly identified repSA gene is essential for pSAM2 replication, encoding a key protein similar to RCR initiator proteins.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- pSAM2 is an 11-kilobase integrating element from Streptomyces ambofaciens.
- Previous studies indicated pSAM2 replicates via a rolling-circle replication (RCR) mechanism, generating single-stranded DNA.
- Replication involves two distinct regions, one containing the plus origin of replication (ds origin).
Purpose of the Study:
- To investigate the second region involved in pSAM2 replication.
- To identify and characterize open reading frames (ORFs) within this region.
- To determine the function of identified ORFs in pSAM2 replication.
Main Methods:
- Nucleotide sequencing of the second replication region.
- Bioinformatic analysis to identify putative ORFs.
- Functional analysis of identified ORFs, including replication assays in Streptomyces.
Main Results:
- Three putative ORFs were identified: orf183, orf50, and repSA (encoding 183, 50, and 459 amino acids, respectively).
- orf183 is not essential for replication, and the function of orf50 remains unknown.
- repSA is essential for pSAM2 replication and encodes a protein (RepSA) with similarities to RCR initiator proteins. A construct containing repSA and the ds origin replicated in Streptomyces.
Conclusions:
- The repSA gene encodes the major replication protein of pSAM2.
- RepSA functions as a replication initiator protein for pSAM2, consistent with an RCR mechanism.
- repSA may be co-transcribed with genes involved in pSAM2 integration and excision.