Single-stranded DNA binding properties of the UvsX recombinase of bacteriophage T4: binding parameters and effects of nucleotides 1 1Edited by M. Gottesman

Ando, Richard A.; Morrical, Scott W.

doi:10.1006/jmbi.1998.2124

Cited by 33 publications

(76 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The induction of a stretched DNA conformation is found in nucleoprotein filaments of ssDNA-binding proteins gp32 from phage T4 (17), SSB from E. coli (38), and RPA from yeast (24). This extended conformation is a property shared with protein-DNA filaments formed by recombinases such as bacterial RecA (39), UvsX from phage T4 (25), and eucaryote Rad51 (27). Although this fluorescence increase does not depend in a quantitative fashion on DNA length, it is nevertheless a useful qualitative manner to compare conformational changes brought about by various ssDNA-binding proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Larger fluorescence enhancement values are reported for active recombinases in the presence of ATP cofactor at 25°C: UvsX protein (2.0 -2.5 (25)); RecA protein (2.8 (21, 26)); and Rad51 at 30°C (5.0 (27)). There have been reports that the fluorescence enhancement of ⑀DNA varies with the quantity and quality of etheno modification (25). We therefore compared the fluorescence enhancement of ICP8 and RecA using the same ⑀DNA substrate.…”

Section: Resultsmentioning

confidence: 99%

“…Recombination proficient nucleoprotein filaments formed by UvsX (25), RecA (26), and Rad51 (27) are stable in the presence of NaCl, whereas inactive recombinase filaments (such as RecA protein in the presence of ADP) are readily disrupted by salts (39).…”

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

Equilibrium Binding of Single-stranded DNA with Herpes Simplex Virus Type I-coded Single-stranded DNA-binding Protein, ICP8

Gourves¹,

Gac²,

Villani³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have carried out solution equilibrium binding studies of ICP8, the major single-stranded DNA (ssDNA)-binding protein of herpes simplex virus type I, in order to determine the thermodynamic parameters for its interaction with ssDNA. Fluorescence anisotropy measurements of a 5-fluorescein-labeled 32-mer oligonucleotide revealed that ICP8 formed a nucleoprotein filament on ssDNA with a binding site size of 10 nucleotides/ICP8 monomer, an association constant at 25°C, K ‫؍‬ 0.55 ؎ 0.05 ؋ 10 6 M ؊1, and a cooperativity parameter, ‫؍‬ 15 ؎ 3. The equilibrium constant was largely independent of salt, ␦log(K)/␦log([NaCl]) ‫؍‬ ؊2.4 ؎ 0.4. Comparison of these parameters with other ssDNA-binding proteins showed that ICP8 reacted with an unusual mechanism characterized by low cooperativity and weak binding. In addition, the reaction product was more stable at high salt concentrations, and fluorescence enhancement of etheno-ssDNA by ICP8 was higher than for other ssDNA-binding proteins. These last two characteristics are also found for protein-DNA complexes formed by recombinases in their active conformation. Given the proposed role of ICP8 in promoting strand transfer reactions, they suggest that ICP8 and recombinase proteins may catalyze homologous recombination by a similar mechanism.ICP8 is 128-kDa zinc metalloprotein coded by the UL29 gene of herpes simplex virus type 1 (HSV-1).1 It is one of seven virus-encoded proteins necessary for HSV-1 origin-dependent DNA replication (1). ICP8 forms an extended nucleoprotein filament with ssDNA in vitro; it binds more readily to ssDNA than to double-stranded DNA (1-6), and DNA binding may involve contacts by free sulfhydryl groups and surface lysine and tyrosine residues (6, 7). In addition to its role as an ssDNAbinding protein, complexes of ICP8 with other proteins may act during viral replication. ICP8 physically interacts via the UL8 protein with the HSV-1 helicase primase heterotrimer thereby stimulating unwinding of intact and damaged duplex DNA (8, 9); likewise a physical interaction between ICP8 and the herpes UL9 helicase has also been shown (10). Specific protein-protein interactions between ICP8, UL9 protein, and subunits of the DNA helicase primase are required for the assembly of these proteins into prereplicative sites, and recruitment of the DNA polymerase into this complex is mediated by the UL42 subunit (11,12).Cells infected with HSV-1 display a high frequency of homologous recombination (13). In vitro experiments suggest that ICP8 may be responsible for homologous recombination in vivo. ICP8 exhibits helix destabilizing and DNA renaturing activities (2, 14). The protein also catalyzes homologous pairing and strand exchange activity in vitro (15) where it is able to transfer a DNA strand from a linear duplex to a complementary single-stranded circular DNA. The reaction requires MgCl 2 but not (d)NTPs, and strand exchange is limited to a few 100 base pairs.Understanding the role of ICP8 in the DNA replication and recombination reactions of HSV-1 will require kn...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Equilibrium Binding of Single-stranded DNA with Herpes Simplex Virus Type I-coded Single-stranded DNA-binding Protein, ICP8

Gourves¹,

Gac²,

Villani³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Most of these genes were first identified by the isolation of amber or temperature-sensitive conditional-lethal mutations and were assigned numbers (Table 2) before their functions were determined (264). 5,6,7,8,9,10,11,12,15,18,19,25,26,27,28,29,48,53 a Genes in parentheses appear in another, primary category. Primary functional assignments and the corresponding colors are used in Fig.…”

Section: Characterized T4 Genes and The Early Geneticsmentioning

confidence: 99%

Bacteriophage T4 Genome

Miller

Kutter

Mosig

et al. 2003

Microbiol Mol Biol Rev

704

801

View full text Add to dashboard Cite

SUMMARY Phage T4 has provided countless contributions to the paradigms of genetics and biochemistry. Its complete genome sequence of 168,903 bp encodes about 300 gene products. T4 biology and its genomic sequence provide the best-understood model for modern functional genomics and proteomics. Variations on gene expression, including overlapping genes, internal translation initiation, spliced genes, translational bypassing, and RNA processing, alert us to the caveats of purely computational methods. The T4 transcriptional pattern reflects its dependence on the host RNA polymerase and the use of phage-encoded proteins that sequentially modify RNA polymerase; transcriptional activator proteins, a phage sigma factor, anti-sigma, and sigma decoy proteins also act to specify early, middle, and late promoter recognition. Posttranscriptional controls by T4 provide excellent systems for the study of RNA-dependent processes, particularly at the structural level. The redundancy of DNA replication and recombination systems of T4 reveals how phage and other genomes are stably replicated and repaired in different environments, providing insight into genome evolution and adaptations to new hosts and growth environments. Moreover, genomic sequence analysis has provided new insights into tail fiber variation, lysis, gene duplications, and membrane localization of proteins, while high-resolution structural determination of the “cell-puncturing device,” combined with the three-dimensional image reconstruction of the baseplate, has revealed the mechanism of penetration during infection. Despite these advances, nearly 130 potential T4 genes remain uncharacterized. Current phage-sequencing initiatives are now revealing the similarities and differences among members of the T4 family, including those that infect bacteria other than Escherichia coli. T4 functional genomics will aid in the interpretation of these newly sequenced T4-related genomes and in broadening our understanding of the complex evolution and ecology of phages—the most abundant and among the most ancient biological entities on Earth.

show abstract

“…would greatly increase the probability of uvsX protein locally displacing gp32 to nucleate presynaptic filament formation (12,13).…”

Section: Role Of Uvsy Protein In Assembly Of the T4 Presynaptic Filamentmentioning

confidence: 99%

Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Bleuit

et al. 2001

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

Self Cite

View full text Add to dashboard Cite

Studies of recombination-dependent replication (RDR) in the T4system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3 ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop͞replication fork. This partitioning mechanism enforced by the T4 recombination͞replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX͞uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative ''bubble migration'' mode of RDR could play a role in lesion bypass by the T4 replication machinery. Bacteriophage T4 provides an excellent model system for biochemical and genetic studies of recombinationdependent replication (RDR), because DNA replication and recombination are closely coupled throughout much of the phage life cycle. After infecting a host Escherichia coli cell, T4 first replicates its genome via an origin-dependent replication initiation pathway. This pathway is shut off after a few rounds of replication, after expression of the T4 uvsW RNA͞DNA helicase, which resolves R loops required for origin function (1). T4 then relies on a recombination-dependent mechanism to initiate DNA synthesis, and this pathway accounts for a large fraction of the total DNA synthesis observed during T4 infection. In the T4 RDR pathway (reviewed in refs. 2 and 3), branched recombination intermediates generated by the phage homologous recombination machinery are captured and converted into semiconservative DNA replication forks. T4 RDR requires all of the major phage-encoded DNA replication and recombination enzymes including: gp43 (DNA polymerase), gp45 (sliding clamp), gp44͞62 (clamp loader), gp32 [single-stranded DNA (ssDNA) binding protein or ssb], gp61 (primase), gp41 (DNA helicase), gp59 (helicase loader; replication mediator protein or RMP...

show abstract

Single-stranded DNA binding properties of the UvsX recombinase of bacteriophage T4: binding parameters and effects of nucleotides 1 1Edited by M. Gottesman

Cited by 33 publications

References 49 publications

Equilibrium Binding of Single-stranded DNA with Herpes Simplex Virus Type I-coded Single-stranded DNA-binding Protein, ICP8

Equilibrium Binding of Single-stranded DNA with Herpes Simplex Virus Type I-coded Single-stranded DNA-binding Protein, ICP8

Bacteriophage T4 Genome

Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Contact Info

Product

Resources

About