Initiation of bacteriophage phi 29 DNA replication in vivo: Assembly of a membrane-associated multiprotein complex

被引:38
作者
Bravo, A [1 ]
Salas, M [1 ]
机构
[1] AUTONOMOUS UNIV MADRID,CTR BIOL MOL SEVERO OCHOA,CSIC,E-28049 MADRID,SPAIN
基金
美国国家卫生研究院;
关键词
DNA replication; membrane-associated proteins; terminal protein; phage phi 29; Bacillus subtilis; SUBTILIS PHAGE PHI-29; SUPPRESSOR-SENSITIVE MUTANTS; BACILLUS-SUBTILIS; NUCLEAR MATRIX; VIRAL PROTEIN-P6; ADENOVIRUS DNA; INVITRO; TRANSCRIPTION; ELONGATION; TEMPLATES;
D O I
10.1006/jmbi.1997.1032
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Initiation of in vitro phage empty set29 DNA replication requires the formation of a heterodimer between a free molecule of terminal protein (TP), which acts as primer, and the viral DNA polymerase. We have analyzed membrane vesicles from empty set29-infected Bacillus subtilis cells by quantitative immunoblot techniques. During phage DNA synthesis, large amounts of the viral proteins p1 and free TP were recovered in membrane fractions, as well as a low percentage of the total viral DNA polymerase. Interestingly, the amount of DNA polymerase in membrane fractions increased when viral DNA replication was blocked. Both protein p1 and free TP showed affinity for membranes in the absence of viral DNA. The association of protein p1 with membranes was abolished when the C-terminal 43 amino acid residues were deleted. The above results, together with the critical role of protein p1 for in vivo empty set29 DNA replication, led us to conclude that a preliminary stage in the initiation of in vivo empty set29 DNA replication could be the assembly of a membrane-associated multiprotein complex containing at least protein p1, free TP and DNA polymerase. Membrane-attachment of this complex could be directly mediated by both protein p1 and free TP. The ability of free TP to bind to membranes and to prime empty set29 DNA replication would enable a nascent viral DNA molecule to become membrane-associated when its synthesis begins. We postulate that a general function of the TPs covalently linked to linear DNA genomes in prokaryotes might be, in addition to act as primer, to anchor the linear DNA molecule to the bacterial membrane. (C) 1997 Academic Press Limited.
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页码:102 / 112
页数:11
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