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Andrea Berman Center for Structural Biology Phone:
(203) 432-5795 Email: berman {at] csb.yale.edu |
Structural Studies of Terminal Protein-primed DNA replication by Phi 29 DNA polymerase
DNA synthesis usually requires a preexisting
oligonucleotide primer for initiation. However, DNA polymerases, encoded
by viruses such as hepatitis and human adenovirus, uniquely initiate
replication from a “terminal protein” and prime synthesis from a nucleotide
they covalently attach to the terminal protein. The Phi 29 bacteriophage
of B. subtilis has the most biochemically well-characterized
protein-primed replication system; therefore, it can serve as a model for the
more complex human viruses. Although the initiation, transition, and
elongation steps of Phi 29 DNA replication have been extensively biochemically
characterized by the laboratory of Dr. Margarita Salas (Centro de Biologia
Molecular (SCIC-UAM), Universidad Autonoma, Canto Blanco, Madrid, Spain), none
of these steps have been structurally characterized at the atomic level.
Therefore, we (Satwik
Kamtekar and I) propose using X-ray crystallography to visualize the steps
of Phi 29 DNA replication. Analysis of these novel structures will
further our understanding of protein priming in Phi29, lending insight into the
replication of related disease-causing viruses.
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Initiation, transition, and elongation stages occurring at each origin of replication of the 20 kb genome of Bacteriophage Phi 29
Primed by TP, the process of initiation begins with the deoxynucleotidylation of TP Ser232 by Phi 29 pol [1-4]. The second nucleotide, rather than the terminal nucleotide, of the template directs the addition of dATP to TP forming TP-dAMP, the initiation product. In order to recover the information in the 3’ nucleotide of the template, the TP-dAMP complex undergoes an asymmetric translocation (sliding-back), pairing the dAMP with the 3’ terminal nucleotide of the template; the terminal repetition (3’TT) is crucial for this “sliding-back” mechanism to occur [5]. The free 3’OH of the Phi 29 pol-TP-dAMP complex then serves as the primer for the addition of four nucleotides. This second priming event is unique among replicative polymerases. However, before Phi 29 pol can replicate the entire genome, it must dissociate from the 5’ bound primer TP. Biochemical evidence suggests that a structural transition, resulting in this dissociation, occurs during the addition of nucleotides 6-9 [6]. |
References :
1. Mellado, R. P., Penalva, M. A., Inciarte, M. R., Salas M. (1980). The Protein Covalently Linked to the 5' Termini of the DNA
of Bacillus subtilis Phage Phi 29 is Involved in the Initiation of DNA
Replication. Virology. 104,
84-96.
2. Blanco, L., Garcia, J. A., Penalva, M. A., Salas M. (1983). Factors involved in the initiation of phage Phi 29 DNA
replication in vitro: requirement of the gene 2 product for the formation of
the protein
p3-dAMP complex. Nucleic
Acids Res. 11, 1309-1323.
3. Blanco, L., Salas, M. (1984). Characterization and purification of a
phage Phi 29-encoded DNA polymerase required for the initiation of
replication. Proc Natl Acad Sci
U S A. 81, 5325-5329.
4. Penalva, M. A., Salas, M.
(1982). Initiation of phage
Phi 29 DNA replication in vitro: formation of a covalent complex between the
terminal protein, p3, and 5'-dAMP. Proc Natl Acad Sci U S A. 79,
5522-5526.
5. Mendez, J., Blanco, L., Esteban, J. A., Bernad, A., Salas, M. (1992). Initiation of Phi 29 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 U S A. 89, 9579-9583.
6. Mendez, J., Blanco, L., Salas, M.
(1997). Protein-primed DNA
replication: a transition between two modes of priming by a unique DNA
polymerase. EMBO J. 16,
2519-2527.
Publications:
Kamtekar, S.*, Berman,
A.J.*, Wang, J., Lazaro, J.M., de Vega, M., Blanco, L., Salas, M., Steitz,
T.A. (2006). The phi29 DNA
polymerase:protein-primer structure suggests a model for the initiation to
elongation transition. EMBO J. Published online 2 March
2006. PDBID: 2EX3.
Eakin, C.M., Berman, A.J., Miranker, A.D. (2006). A native to amyloidogenic transition regulated by a backbone trigger. Nat Struct Mol Biol. 13, 202-208. PDBID: 2F8O.
Rodriguez, I.,
Lazaro, J.M., Blanco, L., Kamtekar, S., Berman, A.J., Wang, J., Steitz,
T.A., Salas, M., de Vega, M. (2005). A specific subdomain in phi29 DNA polymerase confers both
processivity and strand-displacement capacity. Proc Natl Acad
Sci U S A. 102,
6407-12.
Wang, J., Kamtekar,
S., Berman, A.J., Steitz, T.A.
(2004). Correction of X-ray intensities from single crystals
containing lattice-translocation defects.
Acta Crystallogr D Biol Crystallogr. 61, 67-74.
Kamtekar, S.*, Berman, A.J.*,
Wang, J., Lazaro, J.M., de Vega, M., Blanco, L., Salas, M., Steitz, T.A. (2004) Insights into strand
displacement and processivity from the crystal structure of the protein-primed
DNA polymerase of bacteriophage phi29.
Mol Cell. 16, 1035-6. PDBID: 1XHX, 1XHZ, 1XI1.
Last updated 03/22/2006.
Undergraduate research: Dr. Ealick's Lab at Cornell
University