DNA Topology and the Initiation of Virus DNA Packaging.

Choon Seok Oh,Olga Esther Mejia-Romero,Michael Barton,Priyal Patel,Jean Sippy,Rachel Sippy,Jennifer Crow Hutchinson,Michael Feiss,Bridget Charbonneau,Valentin V Rybenkov

doi:10.1371/journal.pone.0154785

Abstract

During progeny assembly, viruses selectively package virion genomes from a nucleic acid pool that includes host nucleic acids. For large dsDNA viruses, including tailed bacteriophages and herpesviruses, immature viral DNA is recognized and translocated into a preformed icosahedral shell, the prohead. Recognition involves specific interactions between the viral packaging enzyme, terminase, and viral DNA recognition sites. Generally, viral DNA is recognized by terminase’s small subunit (TerS). The large terminase subunit (TerL) contains translocation ATPase and endonuclease domains. In phage lambda, TerS binds a sequence repeated three times in cosB, the recognition site. TerS binding to cosB positions TerL to cut the concatemeric DNA at the adjacent nicking site, cosN. TerL introduces staggered nicks in cosN, generating twelve bp cohesive ends. Terminase separates the cohesive ends and remains bound to the cosB-containing end, in a nucleoprotein structure called Complex I. Complex I docks on the prohead’s portal vertex and translocation ensues. DNA topology plays a role in the TerSλ-cosBλ interaction. Here we show that a site, I2, located between cosN and cosB, is critically important for an early DNA packaging step. I2 contains a complex static bend. I2 mutations block DNA packaging. I2 mutant DNA is cut by terminase at cosN in vitro, but in vivo, no cos cleavage is detected, nor is there evidence for Complex I. Models for what packaging step might be blocked by I2 mutations are presented.

Highlights

Large dsDNA viruses use an ATP hydrolysis-powered motor to package DNA into preformed empty shells, called proheads
To ask if the sequence between cosN and R3 is required for DNA packaging, we constructed a cosmid with an I2 substitution mutation, I2re18-50, in which the 33 bp from λ bp 18 to 50 were changed
The present results show that, in addition to providing proper spacing between cosN and cosB, I2 plays a functional role that is critical for an early step in DNA packaging by λ

Summary

Introduction

Large dsDNA viruses use an ATP hydrolysis-powered motor to package DNA into preformed empty shells, called proheads (reviewed in [1,2,3,4]). An endonucleolytic cut is made to generate the DNA end which is translocated into the prohead to initiate DNA packaging. Many phages, including P22, Sf6, SPP1, and T4 use a headful packaging strategy, in which the initial cut is specific, but subsequent, non-specific cuts are triggered when the prohead is full [5,6,7]. Terminases act processively, such that after the downstream cut, terminase remains bound to the newly-created end in a complex that binds a naive prohead, and sponsors packaging of the chromosome along the concatemer

Methods

Results

Conclusion