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PubMed · 8594371

Using macromolecular crowding agents to identify weak interactions within DNA replication complexes.

M K Reddy·S E Weitzel·S S Daube·T C Jarvis·P H von Hippel
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Bacteriophage T4Chromatography, AffinityDNA LigasesDNA ReplicationDNA, Single-StrandedDNA-Directed DNA PolymeraseEscherichia coliPlasmidsPolyethylene GlycolsPolynucleotide 5'-Hydroxyl-KinaseRec A RecombinasesReplication OriginViral ProteinsJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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M K Reddy, S E Weitzel, S S Daube, T C Jarvis, P H von Hippel. 1995. Using macromolecular crowding agents to identify weak interactions within DNA replication complexes.. https://doi.org/10.1016/0076-6879(95)62038-9

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PubMed1998

The bacteriophage T4 DNA packaging apparatus targets the unexpanded prohead.

During the morphogenesis of the bacteriophage T4 capsid, a conformational change of the major head shell protein, gene product (gp) 23, causes a 50% increase in capsid volume. This expansion is required to accept the full length chromosome and, therefore, must precede the completion of packaging. The expanded shell is thinner and more stable than its precursor, and can bind accessory proteins which further stabilize it. In phages lambda, T3, T7 and P22, expansion occurs during DNA packaging. However, in T4, expanded capsids can package DNA in vitro and expansion occurs in cells infected with packaging-defective mutants, raising the possibility that expansion and packaging are not coupled. Proteolytically mature gp23 (gp23*) in unexpanded proheads is sensitive to chymotrypsin cleavage at Phe154-Ser155, creating a 38 kDa peptide, while gp23* in expanded capsids is refractory to the protease. We used an expansion assay based on this protease sensitivity to determine the expansion status of capsids isolated from various packaging-defective mutants with the goal of determining whether packaging and expansion are normally linked. In infections at 20 degrees C, mutants in the packaging enzymes gp16 and gp17 fail to expand. However, in gene 49(-) mutants, which initiate packaging but fail to complete it, expansion is complete. Thus, packaging drives expansion, and the unexpanded prohead is the substrate for the packaging reaction. We also show that expansion observed in 16(-) and 17(-) infections at 37 degrees C is linked to aberrant packaging. Capsids produced at 15 minutes, when no packaging can be detected, never expand. However, by 35 minutes when aberrant packaging begins, so does expansion of freshly made capsids. Thus in all cases now examined, expansion is only observed in vivo when DNA packaging is also occurring, indicating that these two processes are coupled.

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PubMed1998

Capsid expansion follows the initiation of DNA packaging in bacteriophage T4.

Most bacteriophages undergo a dramatic expansion of their capsids during morphogenesis. In phages lambda, T3, T7 and P22, it has been shown that expansion occurs during the packaging of DNA into the capsid. The terminase-DNA complex docks with the portal vertex of an unexpanded prohead and begins packaging. After some of the DNA has entered, the major head protein undergoes a conformational change that increases both the volume and stability of the capsid. In phage T4, the link between packaging and expansion has not been established. We explored the possibility of such a connection using a pulse-chase protocol and high resolution sucrose gradient analysis of capsid intermediates isolated from wild-type T4-infected cells. We show that the first particle appearing after the pulse is an unexpanded prohead, which can be isolated in vitro as the ESP (empty small particle). The next intermediate to appear is also unexpanded, but contains DNA. This new intermediate, the ISP (initiated small particle), can also be isolated on agarose gels, permitting confirmation of both its expansion state and DNA content ( approximately 10 kbp). It appears, therefore, that >/=8% of the T4 genome enters the head shell prior to expansion. Following packaging of an undetermined amount of DNA, the capsid expands, producing the ILP (initiated large particle), which is finally converted to a full head upon the completion of packaging. An expanded, empty prohead, the ELP (empty large particle), was also observed during 37 degrees C infections, but failed to mature to phage during the chase. Thus the ELP is unlikely to be an intermediate in normal head assembly. We conclude by suggesting that studies on assembly benefit from an emphasis on the processes involved, rather than on the structural intermediates which accumulate if these processes are interrupted.

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