φ29 DNA Polymerase Residue Phe128 of the Highly Conserved (S/T)Lx2h Motif is Required for a Stable and Functional Interaction with the Terminal Protein
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- 作者：Rodrí ; guez ; Irene ; Lá ; zaro ; José ; M. ; Salas ; Margarita ; Vega ; Miguel de
- 关键词：DNA polymerase ; site-directed mutagenesis ; linear DNA replication ; protein-priming ; terminal protein ; Pol I ; E.coli DNA polymerase I ; Pol Ik ; Klenow fragment of Pol I ; ssDNA ; single-stranded DNA ; dsDNA ; double-stranded DNA
- 刊名：Journal of Molecular Biology
- 出版年：2003
- 出版时间：January 3, 2003
- 年：2003
- 卷：325
- 期：1
- 页码：85-97
- 全文大小：987 K
文摘
Bacteriophage φ29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as primer for initiation of DNA replication. By multiple sequence alignments of DNA polymerases from such a family, we have been able to identify two amino acid residues specifically conserved in the protein-priming subgroup of DNA polymerases, a phenylalanine contained in the (S/T)Lx2h motif, and a glutamate belonging to the Exo III motif. Here, we have studied the functional role of these residues in reactions that are specific for DNA polymerases that use a protein-primed DNA replication mechanism, by site-directed mutagenesis in the corresponding amino acid residues, Phe128 and Glu161 of φ29 DNA polymerase. Mutations introduced at residue Phe128 severely impaired the protein-primed replication capacity of the polymerase, being the interaction with the terminal protein (TP) moderately (mutant F128A) or severely (mutant F128Y) diminished. As a consequence, very few initiation products were obtained, and essentially no transition products were detected. Interestingly, φ29 DNA polymerase mutant F128Y showed a decreased binding affinity for short template DNA molecules. These results, together with the high degree of conservation of Phe128 residue among protein-primed DNA polymerases, suggest a functional role for this amino acid residue in making contacts with the TP during the first steps of genome replication and with DNA in the further replication steps.