Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins

Gascón, Irene; Lázaro, José Portolés; Salas, Margarita

doi:10.1093/nar/28.10.2034

Cited by 25 publications

(31 citation statements)

References 50 publications

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“…6, incubation in the absence of protein did not result in release of the labeled oligonucleotide, indicating that the hybrid substrate was stable throughout the experiment. In agreement with previously published results (17,24), the 29 SSB p5 was able to displace the labeled oligonucleotide from the M13 DNA. However, no displacement of the oligonucleotide was detected when the hybrid substrate was incubated with protein p16.7A up to a concentration of 112 M. To facilitate the oligonucleotide displacement, these assays were also performed using an oligonucleotide that contained, in addition to the 17 complementary nucleotides, either a non-complementary 5Ј-(5 thymidines) or a 3Ј-(5 adenines) tail.…”

Section: Protein P167a Has No Helix-destabilizing Activity and Has Nsupporting

confidence: 93%

“…The 29 DNA polymerase has strong affinity for naked ssDNA (28) but does not bind ssDNA when it is covered with the SSB p5 (17,23). In addition, free 29 DNA polymerase, but not when bound to M13 ssDNA, can degrade a single-stranded oligonucleotide due to its 3Ј-5Ј exonucleolytic activity.…”

Section: Protein P167a Has No Effect On In Vitro 29 Dna Initiation Omentioning

confidence: 99%

“…First, binding of the 29 SSB protects ssDNA against nuclease degradation (25,26). Second, it prevents non-productive binding of the 29 DNA polymerase to ssDNA (17,23). Third, it prevents and removes the formation of DNA secondary structures (23,24).…”

Section: Protein P167a Has No Helix-destabilizing Activity and Has Nmentioning

confidence: 99%

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The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

Serna-Rico¹,

Salas

Meijer³

2002

Journal of Biological Chemistry

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The functional role of the 29-encoded integral membrane protein p16.7 in phage DNA replication was studied using a soluble variant, p16.7A, lacking the N-terminal membrane-spanning domain. Because of the protein-primed mechanism of DNA replication, the bacteriophage 29 replication intermediates contain long stretches of single-stranded DNA (ssDNA Studies on DNA replication and related processes have provided detailed insights in the function of many proteins involved in these processes (for review, see Ref. 1). Despite this, little is known about the in vivo organization of DNA replication. To gain a better insight in this fundamental process, we studied the in vivo DNA replication of the Bacillus subtilis bacteriophage 29 (2). The detailed knowledge of its in vitro mechanism of DNA replication (for reviews, see Refs. 3 and 4) made 29 an attractive system for this study.The genome of 29 is a linear double-stranded DNA (dsDNA) 1 of 19,285 base pairs that contains a terminal protein (TP) covalently linked at each 5Ј end. Fig. 1A shows a schematic representation of the genetic and transcriptional organization of the 29 genome. Regulation of 29 DNA transcription, which can be divided into an early and a late stage, has been studied extensively in vivo as well as in vitro (for reviews, see Refs. 3 and 5). The late expressed genes, all transcribed from a single operon present in the central part of the genome, encode the structural proteins of the phage, proteins involved in morphogenesis, and those required for lysis of the infected cell. The early expressed genes are present in two operons that flank the late operon. The early operon located at the left side of the 29 genome encodes the transcriptional regulator protein p4 and various proteins that are directly involved in phage DNA replication, such as the DNA polymerase, TP, singlestranded DNA-binding protein (SSB), double-stranded DNAbinding protein, and protein p1. The operon located at the right side of the 29 genome encodes, in addition to proteins p17 and p16.7, four putative proteins of unknown function.A schematic overview of the in vitro 29 DNA replication mechanism is shown in Fig. 1B. Initiation of 29 DNA replication occurs via a so-called protein-primed mechanism (reviewed in Refs. 3, 4, and 6). The TP-containing DNA ends constitute the origins of replication. Initiation of DNA replication starts by recognition of the origin by a heterodimer formed by the 29 DNA polymerase and the primer TP. The DNA polymerase then catalyzes the addition of the first dAMP to the primer TP. Next, after a transition step, these two proteins dissociate, and the DNA polymerase continues processive elongation until replication of the nascent DNA strand is completed. Replication, which starts at both DNA ends, is coupled to strand displacement. This results in the generation of socalled type I replication intermediates consisting of full-length double-stranded 29 DNA molecules with one or more singlestranded DNA (ssDNA) branches of varying lengths. When the two converging DNA...

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Section: Protein P167a Has No Helix-destabilizing Activity and Has Nsupporting

confidence: 93%

Section: Protein P167a Has No Effect On In Vitro 29 Dna Initiation Omentioning

confidence: 99%

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The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

Serna-Rico¹,

Salas

Meijer³

2002

Journal of Biological Chemistry

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“…Thus, it binds ssDNA with higher affinity and displays helix-destabilizing ability with lower protein concentrations than the other two SSBs (20,21). The complex formed by GA-1 SSB with ssDNA is clearly dif-ferent from the 29-like nucleoprotein complex, as determined by electron microscopy in the presence of DNA (19,20).…”

mentioning

confidence: 92%

Importance of the N-terminal Region of the Phage GA-1 Single-stranded DNA-binding Protein for Its Self-interaction Ability and Functionality

Gascón¹,

Carrascosa²,

Villar³

et al. 2002

Journal of Biological Chemistry

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The single-stranded DNA-binding protein (SSB) of phage GA-1 displays higher efficiency than the SSBs of the related phages 29 and Nf. In this work, the selfinteraction ability of GA-1 SSB has been analyzed by visualization of the purified protein by electron microscopy, glycerol gradient sedimentation, and in vivo crosslinking of bacterial cultures infected with phage GA-1. GA-1 SSB contains an insert at its N-terminal region that is not present in the SSBs of 29 and Nf. Three deletion mutant proteins have been characterized, ⌬N19, ⌬N26, and ⌬N33, which lack the 19, 26 or 33 amino acids, respectively, that follow the initial methionine of GA-1 SSB. Mutant protein ⌬N19 retains the structural and functional behavior of GA-1 SSB, whereas mutant proteins ⌬N26 and ⌬N33 no longer stimulate viral DNA replication or display helix-destabilizing activity. Analysis of the mutant proteins by ultracentrifugation in glycerol gradients and electron microscopy indicates that deletion of 26 or 33 but not of 19 amino acids of the N-terminal region of GA-1 SSB results in the loss of the oligomerization ability of this protein. Our data support the importance of the N-terminal region of GA-1 SSB for the differential self-interaction ability and functional behavior of this protein.Single-stranded DNA-binding proteins (SSBs) 1 contribute to DNA metabolism, playing essential roles in different processes such as DNA replication, repair, and recombination (1-3). SSBs bind single-stranded DNA (ssDNA) in a selective, cooperative, and non-sequence-specific way, protecting it from nuclease attack and preventing the formation of secondary structures on it. SSBs are ubiquitous. They have been isolated from bacteria and their phages, eukarya and their viruses, and archaea (1-6).Different oligomerization states have been reported for SSBs. Thus, monomeric (T4gp32 from bacteriophage T4 or AdDBP from adenovirus (5)), dimeric (SSBs from filamentous phages M13 (1) and Pf3 (7)), homotetrameric (EcoSSB from Escherichia coli (2, 8) or human mitochondrial SSB (9)), and heterotrimeric (hRPA, human RPA (3)) SSBs have been described. Important differences have been also reported mainly concerning the amino acid sequence and ssDNA binding properties of the proteins of this family. The function of the SSB is particularly important in the case of organisms that replicate their genetic material via a proteinpriming mechanism followed by strand displacement, as large amounts of ssDNA are generated in the process (10 -12). This is the case of adenovirus and of the 29 family of Bacillus phages. The genome of the latter consists of a linear dsDNA molecule of about 20 kb that contains a phage-encoded terminal protein (TP) covalently linked to each 5Ј end. Replication of the viral genome starts at either DNA end non-simultaneously by a protein-priming mechanism. After a sliding-back step (13), the viral DNA polymerase elongates the initiation product proceeding by strand displacement toward the other DNA end. The displaced strand is cooperatively bound by the viral...

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“…Interestingly, SSB of a ø29-related phage GA-1 has different characteristics from that of ø29. It contains an insert at its N-terminal region which is not present in ø29 gp5, and self-interacts and forms filamentous structures similar to ø29 gp1 (Gascón et al, 2000a(Gascón et al, , b, 2002. Biochemical study using N-terminal deletion mutants of GA-1 SSB showed that the N-terminal insert was important for the self-interaction, the effective binding to ssDNA, and the stimulatory effect on viral DNA replication in vitro (Gascón et al, 2002).…”

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confidence: 99%

Isolation of suppressors of the temperature-sensitive growth caused by a nonsense mutation in gene 1 of <i>Bacillus subtilis</i> phage ø29 using hydroxylamine

Tone

Kinoshita

Hanagata

et al. 2015

J. Gen. Appl. Microbiol.

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Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins

Cited by 25 publications

References 50 publications

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

The Bacillus subtilis Phage φ29 Protein p16.7, Involved in φ29 DNA Replication, Is a Membrane-localized Single-stranded DNA-binding Protein

Importance of the N-terminal Region of the Phage GA-1 Single-stranded DNA-binding Protein for Its Self-interaction Ability and Functionality

Isolation of suppressors of the temperature-sensitive growth caused by a nonsense mutation in gene 1 of <i>Bacillus subtilis</i> phage ø29 using hydroxylamine

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