Self-association Properties of the Bacteriophage λ Terminase Holoenzyme: Implications for the DNA Packaging Motor

Maluf, Nasib K.; Yang, Qin; Catalano, Carlos Enrique

doi:10.1016/j.jmb.2005.01.016

Cited by 59 publications

(119 citation statements)

References 69 publications

(116 reference statements)

Supporting

Mentioning

104

Contrasting

Order By: Relevance

“…The terminase DNA-recognition components of bacteriophages T4, T7, SPP1 and P22 have been reported to form oligomers (12)(13)(14)(15)(16). In bacteriophage lambda, the two components of terminase, gpNu1 and gpA, formed heterooligomers (17). Formation of oligomers has been shown to be critical for the function of the DNA-recognition components (15,18).…”

mentioning

confidence: 99%

Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

Zhao

Finch

Sequeira

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

101

View full text Add to dashboard Cite

In herpesviruses and many bacterial viruses, genome-packaging is a precisely mediated process fulfilled by a virally encoded molecular machine called terminase that consists of two protein components: A DNA-recognition component that defines the specificity for packaged DNA, and a catalytic component that provides energy for the packaging reaction by hydrolyzing ATP. The terminase docks onto the portal protein complex embedded in a single vertex of a preformed viral protein shell called procapsid, and pumps the viral DNA into the procapsid through a conduit formed by the portal. Here we report the 1.65 Å resolution structure of the DNA-recognition component gp1 of the Shigella bacteriophage Sf6 genomepackaging machine. The structure reveals a ring-like octamer formed by interweaved protein monomers with a highly extended fold, embracing a tunnel through which DNA may be translocated. The N-terminal DNA-binding domains form the peripheral appendages surrounding the octamer. The central domain contributes to oligomerization through interactions of bundled helices. The C-terminal domain forms a barrel with parallel beta-strands. The structure reveals a common scheme for oligomerization of terminase DNA-recognition components, and provides insights into the role of gp1 in formation of the packaging-competent terminase complex and assembly of the terminase with the portal, in which ring-like protein oligomers stack together to form a continuous channel for viral DNA translocation.terminase | DNA-packaging | oligomer | bacteriophage | protein:DNA interaction I n many tailed double-stranded DNA (dsDNA) bacteriophages and herpesvirus, the newly synthesized viral DNA in the infected host cell is present as a concatemer that is comprised of multiple copies of unit-length genome. The concatemeric DNA is packaged into a preformed capsid precursor termed procapsid through a channel formed by a portal protein complex embedded in a unique vertex of the procapsid (1-5). The DNApackaging process involves a precisely coordinated sequence of molecular events, driven by a molecular machine called terminase consisting of two proteins: A DNA-recognition component and a catalytic component, also known as the "small" and "large" subunit, respectively. The terminase specifically binds to concatemeric viral DNA through the DNA-recognition component and cleaves it using the catalytic component, generating a new terminus for the DNA. This terminus is threaded through the portal protein channel embedded in the procapsid, and the catalytic component pumps the DNA into the procapsid in an ATP-dependent manner. When an appropriate amount of DNA is inserted, the terminase cuts the DNA again, dissociates from the procapsid, and can start the next cycle of DNA-packaging. Many of these features in DNA-packaging are also shared by adenoviruses (6). The x-ray structures of the catalytic component from bacteriophage T4 and the portals from various phages were previously described (7-10). However, it is not known how the terminases are assembled and h...

show abstract

mentioning

confidence: 99%

Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

Zhao

Finch

Sequeira

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

101

View full text Add to dashboard Cite

show abstract

“…nuclease and packaging domains starts to be understood in the case of phage T4 (10) and (7,8). The recent sequencing of the T5 genome has allowed identification of the genes encoding the different partners of the encapsidation machinery.…”

Section: Discussionmentioning

confidence: 99%

“…Such general organization is well conserved among tailed phages and herpes viruses (6). The functional characterization of these proteins and the interplay of the different domains is the object of intensive investigation, specially in the case of phage (1,(7)(8)(9), T4 (10,11), and SPP1 (12,13). The terminase large subunit is a two-domain protein.…”

mentioning

confidence: 99%

The Endonuclease Domain of Bacteriophage Terminases Belongs to the Resolvase/Integrase/Ribonuclease H Superfamily

Ponchon

Boulanger

Labesse

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.Packaging of the double-stranded DNA of bacteriophages into the viral capsid relies on a molecular machine consisting of several proteins: the portal pore, which is located at one vertex of the capsid, and the terminases. The latter are implicated, first, in bringing the DNA concatemers to the empty prohead through interaction with the portal protein, then in active ATP-driven translocation of the DNA into the capsid, and, finally, in cutting the concatemer to generate the mature viral DNA (1). Terminases are generally described as hetero-multimeric structures consisting of two subunits. The small regulatory subunit binds to the viral DNA, and the large catalytic subunit carries both an endonuclease activity for DNA cleavage and an ATPase activity required for DNA packaging (2-5). Such general organization is well conserved among tailed phages and herpes viruses (6). The functional characterization of these proteins and the interplay of the different domains is the object of intensive investigation, specially in the case of phage (1, 7-9), T4 (10, 11), and SPP1 (12, 13). The terminase large subunit is a two-domain protein. Its N terminus is well characterized both from sequence and functional analysis. It consists of a conserved ATPase catalytic center with clear ATP binding motifs (named Walker A and B) (14). The C-terminal region is less well defined (10,13,15,16). Mutagenesis studies have shown that the endonuclease activity of phage is located within the C-terminal half of the terminase large subunit (gpA; 73 kDa) (17). Several mutations affecting this region, among which one was located on Asp-401, were shown to inactivate the endonuclease activity (16). Phage T4 large subunit terminase (gp17, 70 kDa) exhibits an in vivo endonuclease activity (18). Extensive site-directed mutagenesis has revealed a clust...

show abstract

“…Holoenzymes-We have demonstrated previously that the purified WT terminase holoenzyme partitions into two discrete species, a homogenous protomer composed of one gpA tightly associated with two gpNu1 polypeptides and a heterogenous oligomer predominantly composed of four protomers (the terminase ring species) (8,9); interconversion between the protomer and homogenous tetrameric ring is facile, but slow. Here, the self-association properties of the purified mutant terminase enzymes were characterized using sedimentation velocity analytical ultracentrifugation as described previously (8,9).…”

Section: Structural Features Of Mutant Terminasementioning

confidence: 99%

“…The "terminase enzyme" is the major active component of the packaging motor responsible for DNA translocation. It is composed of multiple heterotrimer units, each containing a large gpA subunit and two small gpNu1 subunits (8). This ATP-powered complex mediates a number of different activities needed to package unit length genomes from concatemeric substrates; these include endonuclease, strand separation, ATP hydrolysis, and DNA translocation catalytic activities.…”

mentioning

confidence: 99%

Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

Tsay

Sippy

delToro

et al. 2010

Journal of Biological Chemistry

View full text Add to dashboard Cite

Many double-stranded DNA viruses employ ATP-driven motors to translocate their genomes into small, preformed viral capsids against large forces resisting confinement. Here, we show via direct single-molecule measurements that a mutation T194M downstream of the Walker B motif in the phage gpA packaging motor causes an 8-fold reduction in translocation velocity without substantially changing processivity or force dependence, whereas the mutation G212S in the putative C (coupling) motif causes a 3-fold reduction in velocity and a 6-fold reduction in processivity. Meanwhile a T194M pseudorevertant (T194V) showed a near restoration of the wild-type dynamics. Structural comparisons and modeling show that these mutations are in a loop-helix-loop region that positions the key residues of the catalytic motifs, Walker B and C, in the ATPase center and is structurally homologous with analogous regions in chromosome transporters and SF2 RNA helicases. Together with recently published studies of SpoIIIE chromosome transporter and Ded1 RNA helicase mutants, these findings suggest the presence of a structurally conserved region that may be a part of the mechanism that determines motor velocity and processivity in several different types of nucleic acid translocases.Viral DNA packaging motors are remarkable biological nanomachines that are capable of packaging chromosomes to near-crystalline density into viral capsids against enormous internal forces (1, 2). Such motors are present in many doublestranded DNA bacteriophages (3), the medically relevant herpesviruses (4), and possibly in poxviruses and adenoviruses (5). The well characterized genetics and biochemistry of the bacteriophage system make it an ideal model system for investigating the structure-function relationships of DNA-packaging machines (6, 7). The "terminase enzyme" is the major active component of the packaging motor responsible for DNA translocation. It is composed of multiple heterotrimer units, each containing a large gpA subunit and two small gpNu1 subunits (8). This ATP-powered complex mediates a number of different activities needed to package unit length genomes from concatemeric substrates; these include endonuclease, strand separation, ATP hydrolysis, and DNA translocation catalytic activities. The terminase enzyme initiates packaging by binding to a cos site in a linear DNA concatemer composed of several viral genomes. Terminase introduces staggered nicks at the downstream cosN site, 12-bp apart to form the left mature genome end. The motor then binds to a procapsid and translocates DNA into the capsid interior. Packaging is terminated when the terminase encounters the next downstream cos site, which signals the right end of the genome; terminase again nicks the duplex, releasing the DNA-filled capsid. Recent evidence indicates that gpA 1 (gpNu1) 2 heterotrimers assemble into a tetrameric ring, which we presume is representative of the DNA maturation and packaging motor complex (9).Our earlier studies of the ATPase center of the packaging motor includ...

show abstract

Self-association Properties of the Bacteriophage λ Terminase Holoenzyme: Implications for the DNA Packaging Motor

Cited by 59 publications

References 69 publications

Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

Crystal structure of the DNA-recognition component of the bacterial virus Sf6 genome-packaging machine

The Endonuclease Domain of Bacteriophage Terminases Belongs to the Resolvase/Integrase/Ribonuclease H Superfamily

Mutations Altering a Structurally Conserved Loop-Helix-Loop Region of a Viral Packaging Motor Change DNA Translocation Velocity and Processivity

Contact Info

Product

Resources

About