Linear Plasmids and Prophages in Gram Negative Bacteria

Stefan Hertwig

Bundesinstitut für Risikobewertung,

Abteilung 4 - Biologische Sicherheit, 12277 Berlin, Germany [email protected] de

1 Incidence of Linear Plasmids in Gram-Negative Bacteria 142

2 Linear Plasmid Prophages with Covalently Closed Ends 142

2.1 Overall Organization of the Genomes 144

3 Protelomerases are Functional Analogues of Lambdoid Phage Integrases and Belong to the Family of Site-Specific Tyrosine Recombinases 145

3.1 Simple In Vitro and In Vivo Assays to Follow Protelomerase Activity . . . 147

3.2 The Telomere Resolution Site (telRL) Maps Adjacent to the Protelomerase Gene 147

3.2.1 The Most Efficient Binding Substrate for TelN is tos 150

3.2.2 Two TelN Monomers Bind to a Segment of ~ 50 bp at telRL 152

3.2.3 TelO is Essential for the Processing of the Telomere Resolution Site 154

3.2.4 The telRL Site is Cleaved by Staggered Cuts Six Base Pairs Apart

Around the Centre of Dyad Symmetry 155

3.2.5 TelN Binds Covalently to the 3'-Phosphoryl of the Cleaved Strands 156

3.3 Protelomerases are also Involved in Linear Plasmid Replication and Maintenance 157

4 Perspectives 160

References 160

Abstract Among the known linear plasmids in Gram-negative bacteria, there are three well-characterized phages (N15, PY54 and 4>KO2) whose prophages replicate as linear plasmids with covalently closed hairpin ends (telomeres). Close to the left telomere, a gene (tel) resides encoding the protelomerase. This tyrosine recombinase-like enzyme has cleaving-joining activity and is responsible for the generation of the telomeres by processing a palindromic telomere resolution site (telRL) located adjacent to the tel gene. The protelomerase exerts its activity as cleaving-joining enzyme in a concerted action. Functional analyses with the N15 protelomerase TelN demonstrated that two TelN molecules recognize telRL and bind to the target site. Within the centre of telRL (telO), the double-stranded DNA is cut by staggered cleavages six base pairs apart centred around the axis of dyad symmetry of the target site. TelN is transiently attached to the 3' ends of the cleaving sites by covalent binding. Then the new phosphodiester bond with the complementary strand is generated in a transesterification step. Protelomerases are multifunctional enzymes. Besides telomere formation, TelN was shown to be implicated in linear plasmid replication and in the regulation of plasmid partitioning.

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