Characterization of the putative replisome organizer of the lactococcal bacteriophage r1t.
Analysis of the nucleotide sequence of the genome of the lactococcal bacteriophage r1t showed that it may encode at least two proteins involved in DNA replication. On the basis of its similarity with the G38P protein encoded by the Bacillus subtilis phage SPP1, the product of orf11 (Pro11) is thought to be involved in the initiation of phage DNA replication. This protein was overexpressed in Lactococcus lactis and partially purified. Gel retardation analysis using various r1t DNA fragments indicates that Pro11 specifically binds to a sequence located within its cognate gene. DNase I footprinting showed that Pro11 protects a stretch of DNA of 47 bp. This region spans four 6-bp short direct repeats, which suggests that the region contains four binding sites for Pro11. 1,10-Phenanthroline-copper footprinting confirmed the protection of the hexamers. An asymmetric protection pattern of each strand was observed, suggesting that Pro11 contacts each DNA strand separately at contiguous hexamers. We propose a model for the binding of Pro11 to its target sites that may account for the torsion strain required for strand opening at the origin of replication.
Zúñiga
Manuel
M
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Franke-Fayard
Blandine
B
Venema
Gerard
G
Kok
Jan
J
Nauta
Arjen
A
D001435:Bacteriophages / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D003300:Copper; D018983:DNA Footprinting; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013294:Lactococcus lactis / Q000821:virology*; D008969:Molecular Sequence Data; D010618:Phenanthrolines; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; D017361:Viral Nonstructural Proteins; D003300:Copper