Phage N15 telomere resolution : target requirements for recognition and processing by the protelomerase

The Escherichia coli prophage N15 exists as a linear DNA molecule with covalently closed ends. Purified N15 protelomerase TelN is the only protein required to convert circular DNA substrates to the linear form with hairpin termini. Within the center of the telomerase occupancy site tos, the target for TelN is the 56-bp telRL consisting of the central 22-bp palindrome telO and two 14-bp flanking inverted sequence repetitions. DNase I footprinting of TelN-telRL complexes shows a segment of ~50 bp protected by TelN. Surface plasmon resonance studies demonstrate that this extended footprint is caused by two TelN molecules bound to telRL. Stable TelN-target DNA complexes are achieved with telRL; however, the additional sequences of tos stabilize the TelN-target complexes. TelO alone is not sufficient for specific stable complex formation. However, processing can occur, i.e. generation of the linear covalently closed DNA. Within the context of telRL, sequences of telO are involved in specific TelN-telRL complex formation, in processing itself, and/or in recognition of the processing site. The sequence of the central (CG)3 within telO that is part of a 14-bp stretch proposed to have Z-DNA conformation is essential for processing but not for formation of specific TelN-telRL complexes. The concerted action of both TelN molecules at the target site is the basis for telomere resolution. Capturing of reaction intermediates demonstrates that TelN binds covalently to the 3'-phosphoryl of the cleaved strands

Bacteriophage P1 Ban protein is a hexameric DNA helicase that interacts with and substitutes for Escherichia coli DnaB

11 p.-9 fig.-1 tab.Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery. We isolated and sequenced the ban gene, puri®ed the product, and analysed the function of Ban protein in vitro and in vivo. Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions. Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells. This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30oC, but not at 42oC. Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately. This indicates that Ban and DnaB are able to interact in vivo. Complementary to these results, we demonstrate the formation of DnaB±Ban hetero-oligomers in vitro by ion exchange chromatography. We discuss the interaction of bacterial proteins and their phageencoded analogues to ful®l functions that are essential to phage and host growth.This work was supported by grants from the European Commission grant QLRT-1999-30634 to R.D.-O. and E.L. R.D.-O. acknowledges support by the `Programa de Grupos Estrategicos' (C.A.M.) 2000±2003 and grant BIO99-0859-CO1 from the Spanish MEC.Peer reviewe

Hexameric RSF1010 helicase RepA: the structural and functional importance of single amino acid residues

In the known monoclinic crystals the 3-dimensional structure of the hexameric, replicative helicase RepA encoded by plasmid RSF1010 shows 6-fold rotational symmetry. In contrast, in the cubic crystal form at 2.55 Å resolution described here RepA has 3-fold symmetry and consists of a trimer of dimers. To study structure–function relationships, a series of repA deletion mutants and mutations yielding single amino acid exchanges were constructed and the respective gene products were analyzed in vivo and in vitro. Hexamerization of RepA occurs via the N-terminus and is required for NTP hydrolysis. The C-terminus is essential both for the interaction with the replication machinery and for the helicase activity. Functional analyses of RepA variants with single amino acid exchanges confirmed most of the predictions that were based on the published 3-dimensional structure. Of the five motifs conserved in family 4 helicases, all residues conserved in RepA and T7 gp4 helicases participate in DNA unwinding. Residues K42, E76, D77, D139 and H178, proposed to play key roles in catalyzing the hydrolysis of NTPs, are essential for RepA activity. Residue H178 of motif H3 couples nucleotide consumption to DNA strand separation

The repA Gene of the Linear Yersinia enterocolitica Prophage PY54 Functions as a Circular Minimal Replicon in Escherichia coli

The Yersinia enterocolitica prophage PY54 replicates as a linear DNA molecule with covalently closed ends. For replication of a circular PY54 minimal replicon that has been derived from a linear minireplicon, two phage-encoded loci are essential in Escherichia coli: (i) the reading frame of the replication initiation gene repA and (ii) its 212-bp origin located within the 3′ portion of repA. The RepA protein acts in trans on the origin since we have physically separated the PY54 origin and repA onto a two-plasmid origin test system. For this trans action, the repA 3′ end carrying the origin is dispensable. Mutagenesis by alanine scan demonstrated that the motifs for primase and for nucleotide binding present in the protein are essential for RepA activity. The replication initiation functions of RepA are replicon specific. The replication initiation proteins DnaA, DnaG, and DnaB of the host are unable to promote origin replication in the presence of mutant RepA proteins that carry single residue exchanges in these motifs. The proposed origins of the known related hairpin prophages PY54, N15, and PKO2 are all located toward the 3′ end of the corresponding repA genes, where several structure elements are conserved. Origin function depends on the integrity of these elements

Phage P4 origin-binding domain structure reveals a mechanism for regulation of DNA-binding activity by homo- and heterodimerization of winged helix proteins

The origin-binding domain of the gp protein of phage P4 (P4-OBD) mediates origin recognition and regulation of gp activity by the protein Cnr. We have determined the crystal structure of P4-OBD at 2.95 Å resolution. The structure of P4-OBD is that of a dimer with pseudo twofold symmetry. Each subunit has a winged helix topology with a unique structure among initiator proteins. The only structural homologue of the P4-OBD subunit is the DNA-binding domain of the eukaryotic transcriptional activator Rfx1. Based on this structural alignment, a model for origin recognition by the P4-OBD dimer is suggested. P4-OBD mutations that interfere with Cnr binding locate to the dimer interface, indicating that Cnr acts by disrupting the gp dimer. P4-OBD dimerization is mediated by helices 1 and 3 in both subunits, a mode of winged helix protein dimerization that is reminiscent of that of the eukaryotic transcription factors E2F and DP. This, in turn, suggests that Cnr is also a winged helix protein, a possibility that is supported by previously unreported sequence homologies between Cnr and Rfx1 and homology modelling. Hence, in a mechanism that appears to be conserved from phage to man, the DNA-binding activity of winged helix proteins can be regulated by other winged helix proteins via the versatile use of the winged helix motif as a homo- or heterodimerization scaffold

Phage N15 Telomere Resolution : TARGET REQUIREMENTS FOR RECOGNITION AND PROCESSING BY THE PROTELOMERASE

The Escherichia coli prophage N15 exists as a linear DNA molecule with covalently closed ends. Purified N15 protelomerase TelN is the only protein required to convert circular DNA substrates to the linear form with hairpin termini. Within the center of the telomerase occupancy site tos, the target for TelN is the 56-bp telRL consisting of the central 22-bp palindrome telO and two 14-bp flanking inverted sequence repetitions. DNase I footprinting of TelN-telRL complexes shows a segment of ~50 bp protected by TelN. Surface plasmon resonance studies demonstrate that this extended footprint is caused by two TelN molecules bound to telRL. Stable TelN-target DNA complexes are achieved with telRL; however, the additional sequences of tos stabilize the TelN-target complexes. TelO alone is not sufficient for specific stable complex formation. However, processing can occur, i.e. generation of the linear covalently closed DNA. Within the context of telRL, sequences of telO are involved in specific TelN-telRL complex formation, in processing itself, and/or in recognition of the processing site. The sequence of the central (CG)3 within telO that is part of a 14-bp stretch proposed to have Z-DNA conformation is essential for processing but not for formation of specific TelN-telRL complexes. The concerted action of both TelN molecules at the target site is the basis for telomere resolution. Capturing of reaction intermediates demonstrates that TelN binds covalently to the 3'-phosphoryl of the cleaved strands