Abstract
The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage phi29 is absolutely required for viral DNA replication in vivo. About approximately 95% of the intrinsic tyrosine fluorescence of phi29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the phi29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. phi29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the phi29 SSB fluorescence emission (phiF = 0.067 +/- 0.010), (ii) the fast motions of the tyrosine side chains (phi(short) = 0.14 +/- 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I- indicates that phi29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and omega (cooperativity) defining phi29 SSB-poly(dT) interaction, the effective DNA binding constant, K(eff) = K omega, being largely independent of temperature. Altogether, the fluorescent properties of phi29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA.
Highlights
The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage 29 is absolutely required for viral DNA replication in vivo
B. subtilis phage 29 encodes its own SSB, which is absolutely required for viral DNA replication in vivo. 29 SSB plays its role(s) by binding to the single-stranded DNA (ssDNA) regions of the replicative intermediates produced during strand-displacement 29 DNA replication (18 –20)
Towards the Identification of Residues Involved in Complex Formation—Steady-state fluorescence studies have served to determine that the interaction between 29 SSB and ssDNA is characterized by an unlimited cooperative DNA binding mode ( ϭ 50 – 80 at 25 °C), a relatively low binding affinity (Keff ϭ 1–3 ϫ 105 MϪ1 at 25 °C), a small nucleotide binding size (n ϭ 3– 4 nt), and a net release of a very low number of cations [21]
Summary
The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage 29 is absolutely required for viral DNA replication in vivo. Complexes formed by the association of singlestranded DNA-binding proteins (SSBs) with single-stranded DNA (ssDNA) are of particular interest. Even when the amount of DNA template is high, 29 SSB is important for the in vitro system, increasing several times dNTP incorporation [17, 18] This stimulatory effect is related to binding to the ssDNA produced during 29 DNA replication [19], which, likely, protects it from nuclease degradation and prevents unproductive binding of the 29 DNA polymerase. If the three tyrosines of 29 SSB (at positions 50, 57, and 76) were forming part of
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