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

Abstract

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...

Unlimited cooperativity ofBetatectivirusSSB, a novel DNA binding protein related to an atypical group of SSBs from protein-primed replicating bacterial viruses

“…In line with the results of the Y2H Bam35 intraviral interactome [65], where no self-interaction of this protein was observed, B35SSB is a monomer in solution. The podoviral Φ29SSB and Nf SSBs are also monomers but GA-1 SSB is generally a hexamer in solution, although at high salt concentration (200 mM NaCl) it is detected as a monomer [66]. Although a similar salt concentration was used in our ultracentrifugation assays, a lower concentration was used in the crosslinking assays, confirming the monomeric state of B35SSB regardless of the salt concentration.…”

“…In some cases, such as GA-1 and T4 SSBs, the N-terminal domain has a role in cooperativity through self-association mediated by electrostatic interactions [66,69]. Nevertheless, within the C-terminal region of B35SSB, we have identified two conserved positively charged residues, K130 and K156, whose non-conservative mutation severely affects the ssDNA-binding ability.…”

DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

“…Φ29 protein p5 is a single-stranded DNA binding protein (Martin et al, 1989 ) that protects DNA from nucleases (Martin et al, 1989 ) and prevents unproductive binding of Φ29 DNAP to ssDNA generated during replication (Gutiérrez et al, 1991 ). Φ29 SSB has high sequence similarity with SSBs from the related podoviruses Nf and GA-1, although Φ29 and Nf are monomeric in solution, whereas GA-1 SSB is hexameric (Soengas et al, 1995 ; Gascón et al, 2000a ), by means of a N-terminal additional motif (Gascón et al, 2002 ). Podoviral SSBs share some key hydrophobic residues with unrelated viral SSBs (Gutiérrez et al, 1991 ) and, indeed, they may also share the SSBs common OB-fold, as found by secondary structure prediction and multiple sequence alignment (Figure 9 ).…”

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

“…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). In the N-terminal region of GA-1 SSB, a KDYIKVY sequence was especially important because the deletion of this region diminished the functionality of the GA-1 SSB (Gascón et al, 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). In the N-terminal region of GA-1 SSB, a KDYIKVY sequence was especially important because the deletion of this region diminished the functionality of the GA-1 SSB (Gascón et al, 2002). We found that the KDYIKVY sequence is almost perfectly conserved in the C-terminal region of ø29 gp1 (Fig.…”