Abstract

φ29 DNA replication starts at both DNA ends by a protein priming mechanism. The formation of the terminal protein-dAMP initiation complex is directed by the second nucleotide from the 3′ end of the template. The transition from protein-primed initiation to normal DNA elongation has been proposed to occur by a sliding-back mechanism that is necessary for maintaining the sequences at the φ29 DNA ends. Structure—function studies have been carried out in the φ29 DNA polymerase. By site-directed mutagenesis of amino acids conserved among distantly related DNA polymerases we have shown that the N-terminal domain of φ29 DNA polymerase contains the 3′–5′ exonuclease activity and the strand-displacement capacity, whereas the C-terminal domain contains the synthetic activities (protein-primed initiation and DNA polymerization). Viral protein p6 stimulates the initiation of φ29 DNA replication. The structure of the protein p6—DNA complex has been determined, as well as the main signals at the φ29 DNA ends recognized by protein p6. The DNA binding domain of protein p6 has been studied. The results indicate that an α-helical structure located in the N-terminal region of protein p6 is involved in DNA binding through the minor groove. The φ29 protein p5 is the single-stranded DNA binding (SSB) protein involved in φ29 DNA replication, by binding to the displaced single-stranded DNA (ssDNA) in the replication intermediates. In addition, protein p5 is able to unwind duplex DNA. The properties of the φ29 SSB—ssDNA complex are described. Using the four viral proteins, terminal protein, DNA polymerase, protein p6 and the SSB protein, it was possible to amplify the 19285-bp φ29 DNA molecule by a factor of 4000 after 1 h of incubation at 30°C. The infectivity of the in vitro amplified DNA was identical to that of φ29 DNA obtained from virions.

References

[1]
Salas
M.
(
1991
)
Protein-priming of DNA replication
Annu. Rev. Bio
 ,
60
,
39
71
.
[2]
Martín
J.M.
Méndez
E.
Salas
M.
(
1989
)
Characterization of phage φ29 protein p5 as a single-stranded DNA binding protein. Function in φ29 DNA-protein p3 replication
Nucleic Acids Res.
 ,
17
,
3663
3672
.
[3]
Gutiérrez
C.
Martín
G.
Sogo
J.M.
Salas
M.
(
1991
)
Mechanism of stimulation of DNA replication by bacteriophage φ29 single-stranded DNA-binding protein p5
J. Biol. Chem.
 ,
266
,
2104
2111
.
[4]
Gutiérrez
C.
Sogo
J.M.
Salas
M.
(
1991
)
Analysis of replicative intermediates produced during bacteriophage φ29 DNA replication in vitro
J. Mol. Biol.
 ,
222
,
983
994
.
[5]
Serrano
M.
Gutiérrez
J.
Prieto
I.
Hermoso
J.M.
Salas
M.
(
1989
)
Signals at the bacteriophage φ29 DNA replication origins required for protein p6 binding and activity
EMBO J.
 ,
8
,
1879
1885
.
[6]
Otero
M.J.
Salas
M.
(
1989
)
Regions at the carboxyl end of bacteriophage φ29 protein p6 required for DNA binding and activity in φ29 DNA replication
Nucleic Acids Res.
 ,
17
,
4567
4577
.
[7]
Salas
M.
Méndez
J.
Esteban
J.A.
Serrano
M.
Gutiérrez
C.
Hermoso
J.M.
Bravo
A.
Soengas
M.S.
Lázaro
J.M.
Blasco
M.A.
Freire
R.
Bernad
A.
Sogo
J.M.
Blanco
L.
(
1993
)
Terminal protein priming of DNA replication: bacteriophage φ29 as a model system
In:
Virus Strategies. Molecular Biology and Morphogenesis
 
Doerfler
W.
Bohm
P.
, Eds), pp
3
19
VCH
,
Weinheim
.
[8]
Méndez
J.
Blanco
L.
Esteban
J.A.
Bernad
A.
Salas
M.
(
1992
)
Initiation of φ29 DNA replication occurs at the second 3′ nucleotide of the linear template: a sliding-back mechanism for protein-primed DNA replication
Proc. Natl. Acad. Sci. USA
 ,
89
,
9579
9583
.
[9]
Caldentey
J.
Blanco
L.
Bamford
D.H.
Salas
M.
(
1993
)
In vitro replication of bacteriophage PRD1 DNA. Characterization of the protein-primed initiation site
Nucleic Acids Res
 ,
21
,
3725
3730
.
[10]
Bernad
A.
Blanco
L.
Lázaro
J.L.
Martín
G.
Salas
M.
(
1989
)
A conserved 3′–5′ exonuclease active site in prokaryotic and eukaryotic DNA polymerases
Cell
 ,
59
,
219
228
.
[11]
Blanco
L.
Bernad
A.
Blasco
M.A.
Salas
M.
(
1991
)
A general structure for DNA-dependent DNA polymerases
Gene
 ,
100
,
27
38
.
[12]
Ollis
D.L.
Brick
R.
Hamlin
R.
Xuong
N.G.
Steitz
T.A.
(
1985
)
Structure of the large fragment of Escherichia coli DNA polymerase I complexed with TMP
Nature
 ,
313
,
762
766
.
[13]
Blanco
L.
Bernad
A.
Salas
M.
(
1992
)
Evidence favouring the hypothesis of a conserved 3′–5′ exonuclease active site in DNA-dependent DNA polymerases
Gene
 ,
112
,
139
144
.
[14]
Soengas
M.S.
Esteban
J.A.
Lázaro
J.M.
Bernad
A.
Blasco
M.A.
Salas
M.
Blanco
L.
(
1992
)
Site-directed mutagenesis at the Exo III motif of φ29 DNA polymerase. Overlapping structural domains for the 3′–5′ exonuclease and strand-displacement activities
EMBO J
 ,
11
,
4227
4237
.
[15]
Freemont
P.S.
Friedman
J.M.
Beese
L.S.
Sanderson
M.R.
Steitz
T.A.
(
1988
)
Cocrystal structure of an editing complex of Klenow fragment with DNA
Proc. Natl. Acad. Sci. USA
 ,
85
,
8924
8928
.
[16]
Derbyshire
V.
Freemont
P.S.
Sanderson
M.R.
Beese
L.S.
Friedman
J.M.
Joyce
C.M.
Steitz
T.A.
(
1988
)
Genetic and crystallographic studies of the 3′,5′-exonucleolytic site of DNA polymerase I
Science
 ,
240
,
199
201
.
[17]
Derbyshire
V.
Grindley
N.D.F.
Joyce
C.M.
(
1991
)
The 3′-5′ exonuclease of DNA polymerase I of Escherichia coli: contribution of each amino acid at the active site to the reaction
EMBO J
 ,
10
,
17
24
.
[18]
Blanco
L.
Salas
M.
(
1995
)
Mutational analysis of φ29 DNA polymerase
Methods Enzymol.
 ,
262
,
283
294
.
[19]
Blasco
M.A.
Lázaro
J.M.
Blanco
L.
Salas
M.
(
1993
)
φ29 DNA polymerase active site. The conserved amino acid motif ‘Kx3NSxYG’ is involved in template-primer binding and dNTP selection
J. Biol. Chem.
 ,
268
,
16763
16770
.
[20]
Blasco
M.A.
Lázaro
J.M.
Blanco
L.
Salas
M.
(
1993
)
φ29 DNA polymerase active site. Residue Asp249 of conserved amino acid motif Dx2SLYP is critical for synthetic activities
J. Biol. Chem.
 ,
268
,
24106
24113
.
[21]
Blanco
L.
Prieto
I.
Gutiérrez
J.
Bernad
A.
Lázaro
J.M.
Hermoso
J.M.
Salas
M.
(
1987
)
Effect of NH+4 ions on φ29 DNA—protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase
J. Virol.
 ,
61
,
3983
3991
.
[22]
Beese
L.S.
Derbyshire
V.
Steitz
T.A.
(
1993
)
Structure of DNA polymerase I Klenow fragment bound to duplex DNA
Science
 ,
260
,
352
355
.
[23]
Bernad
A.
Lázaro
J.M.
Salas
M.
Blanco
L.
(
1990
)
The highly conserved amino acid sequence Tyr-Gly-Asp-Thr-Asp-Ser in α-like DNA polymerases is required by phage φ29 DNA polymerase for protein-primed initiation and polymerization
Proc. Natl. Acad. Sci. USA
 ,
87
,
4610
4614
.
[24]
Blasco
M.A.
Lázaro
J.M.
Bernad
A.
Blanco
L.
Salas
M.
(
1992
)
φ29 DNA polymerase active site: mutants in conserved residues Tyr 254 and Tyr 390 are affected in dNTP binding
J. Biol. Chem.
 ,
267
,
19427
19434
.
[25]
Blasco
M.A.
Esteban
J.A.
Méndez
J.
Blanco
L.
Salas
M.
(
1992
)
Structural and functional studies on φ29 DNA polymerase
Chromosoma
 ,
102
,
32
38
.
[26]
Prieto
I.
Serrano
M.
Lázaro
J.M.
Salas
M.
Hermoso
J.M.
(
1988
)
Interaction of the bacteriophage φ29 protein p6 with double-stranded DNA
Proc. Natl. Acad. Sci. USA
 ,
85
,
314
318
.
[27]
Suck
D.
Lahm
A.
Oefner
C.
(
1988
)
Structure refinement to 2 Å of a nicked DNA oligonucleotide complex with DNase I
Nature
 ,
332
,
464
468
.
[28]
Serrano
M.
Salas
M.
Hermoso
J.M.
(
1990
)
A novel nucleoprotein complex at a replication origin
Science
 ,
248
,
1012
1016
.
[29]
Pastrana
R.
Lázaro
J.M.
Blanco
L.
García
J.A.
Méndez
E.
Salas
M.
(
1985
)
Overproduction and purification of protein p6 of Bacillus subtilis phage φ29: role in the initiation of DNA replication
Nucleic Acids Res
 ,
13
,
3083
3100
.
[30]
Serrano
M.
Salas
M.
Hermoso
J.M.
(
1993
)
Multimeric complexes formed by DNA-binding proteins of low sequence-specificity
Trends Biochem. Sci.
 ,
18
,
202
206
.
[31]
Serrano
M.
Gutiérrez
C.
Salas
M.
Hermoso
J.M.
(
1993
)
Superhelical path of the DNA in the nucleoprotein complex that activates the initiation of phage φ29 DNA replication
J. Mol. Biol.
 ,
230
,
248
259
.
[32]
Otero
M.J.
Lázaro
J.M.
Salas
M.
(
1990
)
Deletions at the N terminus of bacteriophage φ29 protein p6: DNA binding and activity in φ29 DNA replication
Gene
 ,
95
,
25
30
.
[33]
Saier
M.S.
McCaldon
P.
(
1988
)
Statistical and functional analyses of viral and cellular proteins with N-terminal amphiphatic α-helices with large hydrophobic moments: importance to macromolecular recognition and organelle targeting
J. Bacteriol.
 ,
170
,
2296
2300
.
[34]
Bravo
A.
Hermoso
J.M.
Salas
M.
(
1994
)
A genetic approach to identify functional amino acids in protein p6 of Bacillus subtilis phage φ29
Mol. Gen. Genet.
 ,
245
,
529
536
.
[35]
Soengas
M.S.
Esteban
J.A.
Salas
M.
Gutiérrez
C.
(
1994
)
Complex formation between phage φ29 single-stranded DNA binding protein and DNA
J. Mol. Biol.
 ,
239
,
213
226
.
[36]
Blanco
L.
Bernad
A.
Lázaro
J.M.
Martín
G.
Garmendia
C.
Salas
M.
(
1989
)
Highly efficient DNA synthesis by the phage φ29 DNA polymerase. Symmetrical mode of DNA replication
J. Biol. Chem.
 ,
264
,
8935
8940
.
[37]
Kowalczykowski
S.C.
Bear
D.G.
von Hippel
P.H.
(
1981
)
Single-stranded DNA binding proteins
Boger
P.D.
, Ed)
3rd edn.
,
14
, In:
The Enzymes
 , pp
373
444
Academic Press
,
New York, NY
.
[38]
Alma
N.C.M.
Harmsen
B.J.M.
de Jong
E.A.M.
van de Ven
J.
Hilbers
C.W.
(
1983
)
Fluorescence studies of the complex formation between the gene 5 protein of bacteriophage M13 and polynucleotides
J. Mol. Biol.
 ,
163
,
47
62
.
[39]
Chase
J.W.
Williams
K.R.
(
1986
)
Single-stranded DNA binding proteins required for DNA replication
Annu. Rev. Biochem.
 ,
55
,
103
136
.
[40]
Meyer
R.R.
Laine
P.S.
(
1990
)
The single-stranded DNA-binding protein of Escherichia coli
Microbiol. Rev.
 ,
54
,
342
380
.
[41]
Esteban
J.A.
Soengas
M.S.
Salas
M.
Blanco
L.
(
1994
)
J. Biol. Chem.
 ,
269
,
31946
31954
.
[42]
Méndez
J.
Blanco
L.
Lázaro
J.M.
Salas
M.
(
1994
)
J. Biol. Chem.
 ,
269
,
30030
30038
.
[43]
Blasco
M.A.
Méndez
J.
Lázaro
J.M.
Blanco
L.
Salas
M.
(
1995
)
J. Biol. Chem.
 ,
270
,
2735
2740
.
[44]
Freire
R.
Salas
M.
Hermoso
J.M.
(
1994
)
EMBO J
 ,
13
,
4353
4360
.
[45]
Blanco
L.
Lázaro
J.M.
de Vega
M.
Bonnin
A.
Salas
M.
(
1994
)
Proc. Natl. Acad. Sci. USA
 ,
91
,
12198
12202
.

Author notes

1
Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, NY 11724, USA.
2
Cold Spring Harbor Laboratory, P.O. Box 100, Cold Spring Harbor, NY 11724, USA.