Kinetic Analysis of the Endonuclease Activity of Phage λ Terminase:  Assembly of a Catalytically Competent Nicking Complex Is Rate-Limiting

Woods, Liping; Terpening, Christopher; Catalano, Carlos Enrique

doi:10.1021/bi963044m

Cited by 28 publications

(60 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Studies on the ATPase activity of the isolated terminase subunits have similarly revealed that the isolated gpNu1 subunit possesses weak ATPase activity while gpA efficiently hydrolyzes ATP (17,34,40). Consistently, the isolated hexa-His gpNu1 subunit possesses modest ATP hydrolysis activity, whereas hexa-His gpA hydrolyzes ATP at a rate similar to that of the wild-type subunit (Table V).…”

Section: Catalytic Activity Of the Isolated Wild-type And Hexa-histagmentioning

confidence: 72%

“…The lysates were treated with chloroform, clarified, and plated for phage yield on the IHF ϩ strain MF1427. Terminase Activity Assays-The cos cleavage assay was performed as described previously using pAFP1 as a nuclease substrate (6,34). ATPase catalytic activity was examined as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

“…Kinetic Analysis-Steady-state ATPase activity was analyzed using linear regression techniques as described previously (6,34). Only data within the linear portion of the reaction time course were used in the analysis.…”

Section: Methodsmentioning

confidence: 99%

“…Whereas both enzymes exhibit significant activity in this assay, the time course for the hexa-His-tagged enzyme lags slightly behind that of wild-type terminase. We have suggested that assembly of the terminase subunits onto DNA is the rate-limiting step in the cos-cleavage reaction (6,34), and these data suggested that the hexa-His-tagged holoenzyme might have impaired DNA binding interactions. In order to explore more fully this possibility, we examined the cos-cleavage activity of these enzymes in the absence of E. coli integration host factor (IHF).…”

Section: Atpase Activity Of Wild-type and Hexa-his Terminase Holoenzymentioning

confidence: 98%

See 3 more Smart Citations

Cloning, Expression, and Biochemical Characterization of Hexahistidine-tagged Terminase Proteins

Qi¹,

Woods²,

Feiss³

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The terminase enzyme from bacteriophage is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-Histagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexaHis terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexaHis is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies.Terminase enzymes are found in all of the large, tailed double-stranded DNA bacteriophages and are responsible, at least in part, for the insertion of a viral genome into an empty, pre-formed shell or procapsid (1-3). In bacteriophage , the enzyme is composed of large (gpA, 1 73.2 kDa) and small (gpNu1,20.4 kDa) subunits that are isolated as a gpA 1 ⅐gpNu1 2 holoenzyme complex (4 -6). These proteins are an integral part of a series of nucleoprotein intermediates involved in DNA packaging, however, and the subunit stoichiometry in each of these intermediates likely differs (7-9). The preferred packaging substrate in phage consists of a linear concatemer of viral genomes, linked head-to-tail and up to 10 genomes in length (10). The cohesive end site (cos) of the viral genome represents the junction between successive genomes in the concatemer and is the site where the terminase subunits assemble to initiate DNA packaging (11)(12)(13)(14). A model for genome packaging has been proposed as follows (7-9) (see Fig. 1): ...

show abstract

Section: Catalytic Activity Of the Isolated Wild-type And Hexa-histagmentioning

confidence: 72%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Atpase Activity Of Wild-type and Hexa-his Terminase Holoenzymentioning

confidence: 98%

See 2 more Smart Citations

Cloning, Expression, and Biochemical Characterization of Hexahistidine-tagged Terminase Proteins

Qi¹,

Woods²,

Feiss³

et al. 1999

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The observed rate of cos-cleavage is stimulated by ATP, ADP, and GDP, but interestingly, not GTP [22,29,59,85,106]. IHF also stimulates the observed rate two-to threefold, but only when the terminase concentration is limiting [23,85,107]. The reaction kinetics are characterized by a single enzymatic turnover and the data suggest that two terminase protomers are required to efficiently nick the duplex [23,107].…”

Section: Endonuclease (Cos-cleavage) Activitymentioning

confidence: 92%

The terminase enzyme from bacteriophage lambda: a DNA-packaging machine

Catalano

2000

Cellular and Molecular Life Sciences (CMLS)

158

139

View full text Add to dashboard Cite

This review focuses on the biochemical, biophysical, and catalytic properties of terminase, an enzyme involved in bacteriophage lambda genome packaging. The holoenzyme possesses ATPase, DNA strand-separation, and site-specific nuclease activities that work in concert to insert a viral genome into the confines of a performed capsid. Moreover, the terminase subunits are part of a series of nucleoprotein complexes involved in genome packaging, including remarkably stable intermediates that transition to a highly mobile DNA packaging 'machine.' Models for the assembly and interconversion of these complexes are presented. Interactions between the catalytic sites in the enzyme complex, and modulation of these catalytic activities as it relates to the assembly and relative stability of the packaging intermediates are discussed. This ordered progression of nucleoprotein intermediates is a common theme in biology as demonstrated by mechanistic similarities between viral DNA packaging, the initiation of chromosomal replication, and the initiation of transcription. Terminase is thus part of a growing number of examples of biological 'machines' or molecular 'motors.'

show abstract

Biochemical Characterization of Bacteriophage Lambda Genome Packaging in Vitro

2003

View full text Add to dashboard Cite

Bacteriophage lambda has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level. Limited in vitro packaging efficiency has hampered progress toward this end, however. It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids. We describe the construction of a vector that expresses lambda procapsids with a yield that is 40-fold greater than existing systems. Consistent with previous studies, packaging of a mature lambda genome is very inefficient in vitro, with only 4% of the input procapsids utilized. Concatemeric DNA is the preferred packaging substrate in vivo, and procapsids interact with a nucleoprotein complex known as complex I to initiate genome packaging. When complex I is used as a packaging substrate in vitro, capsid utilization is extremely efficient, and 40% of the input DNA is packaged. Finally, we provide evidence for a packaging-stimulated ATPase activity, and kinetically characterize this reaction quantifying the energetic cost of DNA packaging in bacteriophage lambda.

show abstract

Kinetic Analysis of the Endonuclease Activity of Phage λ Terminase: Assembly of a Catalytically Competent Nicking Complex Is Rate-Limiting

Cited by 28 publications

References 36 publications

Cloning, Expression, and Biochemical Characterization of Hexahistidine-tagged Terminase Proteins

Cloning, Expression, and Biochemical Characterization of Hexahistidine-tagged Terminase Proteins

The terminase enzyme from bacteriophage lambda: a DNA-packaging machine

Biochemical Characterization of Bacteriophage Lambda Genome Packaging in Vitro

Contact Info

Product

Resources

About