Tyrosines involved in the activity of φ29 single-stranded DNA binding protein

Abstract

The genome of Bacillus subtilis phage ϕ29 consists of a linear double-stranded DNA with a terminal protein (TP) covalently linked to each 5' end (TP-DNA). ϕ29 DNA polymerase is the enzyme responsible for viral DNA replication, due to its distinctive properties: high processivity and strand displacement capacity, being able to replicate the entire genome without requiring the assistance of processivity or unwinding factors, unlike most replicases. ϕ29 single-stranded DNA binding protein (SSB) is encoded by the viral gene 5 and binds the ssDNA generated in the replication of the ϕ29 TP-DNA. It has been described to stimulate the DNA elongation rate during the DNA replication. Previous studies proposed residues Tyr50, Tyr57 and Tyr76 as ligands of ssDNA. The role of two of these residues has been determined in this work by site-directed mutagenesis. Our results showed that mutant derivative Y57A was unable to bind to ssDNA, to stimulate the DNA elongation and to displace oligonucleotides annealed to M13 ssDNA, whereas mutant Y50A behaved like the wild-type SSB.

Fig 1. Gel mobility shift assay of the wild-type and mutants SSB.

A 5’-labeled DNA fragment heat denatured (216mer) was incubated for 15 minutes with the indicated amounts of ϕ29 SSB wild-type or mutant at 4°C and subjected to non-denaturing gel electrophoresis. The bands corresponding to free DNA and to the SSB/DNA complex were detected by autoradiography. c: control without SSB. * asterisk indicates 5’ -labeled DNA fragment heat denatured (216mer).

Fig 2. ϕ29 TP-DNA amplification.

The assays were carried out as described in Materials and Methods using 3 nM of ϕ29 DNA polymerase, 30 pM of ϕ29 TP-DNA, 6.5 nM of ϕ29 TP, 35 μM of ϕ29 DBP binding protein and (A) increasing amounts of each ϕ29 SSB or (C) 30 μM of each ϕ29 SSB at different times. The reactions were incubated at 30°C for 80 minutes in A and for the indicated times in B. The length and amount of the synthetized DNA was analyzed by 0.7% alkaline agarose gel electrophoresis followed by autoradiography. c: control without SSB (Fig 2A, line 1). Percentage of amplification obtained in A and C (respect to the control without SSB) are represented in B and D, respectively, as mean ± SD corresponding to three independent experiments: wt SSB (black squares), mutant Y50A (white) mutant Y57A (white triangles).

Fig 3. TP-DNA replication with the exonuclease-deficient ϕ29 DNA polymerase mutant.

The assay was carried out as described in Materials and Methods in the presence of 13 nM of ϕ29 DNA polymerase exo- mutant D12A/D66A, 1.6 nM of TP-DNA, 13 nM of TP and 30 μM of SSB. The samples were incubated at 30°C for the indicated times. The size of the replication products was analyzed by 0.7% alkaline agarose gel electrophoresis followed by autoradiography. The position of unit-length TP-DNA is indicated.

Fig 4. Primed M13 ssDNA replication.

The assay was performed as described in Materials and Methods using 60 nM of ϕ29 DNA polymerase, 5 nM of M13-17mer and 30 μM of the different SSBs. Samples were incubated at 30°C for the indicated times and analyzed by 0.7% alkaline agarose gel electrophoresis followed by autoradiography. The position of unit-length M13 DNA is indicated.

Fig 5. Helix destabilizing activity of ϕ29 SSB wild-type and mutants.

The assay was carried out as described in Materials and Methods using 2 nM of M13-17mer and increasing amounts of each SSB being incubated at 37°C for 60 minutes. The samples were fractionated in an 8% polyacrylamide gel followed by autoradiography. 90°C: heat-denatured substrate; c: without SSB. *asterisk indicates 5’ -labeled oligonucleotide 17mer.