Dataset Information

Dual role of ?29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.

ABSTRACT: Resolution of the crystallographic structure of ?29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3' terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of ?29 DNA polymerase, an enzyme that starts linear ?29 DNA replication using a terminal protein (TP) as primer. Our results show that single replacement of ?29 DNA polymerase residue Lys529 by Ala or Glu decreases the stabilisation of the primer-terminus at the polymerisation active site, impairing both the insertion of the incoming nucleotide when DNA and TP are used as primers and the translocation step required for the next incoming nucleotide incorporation. In addition, combination of the DNA polymerase mutants with a TP derivative at residue Glu233, neighbour to the priming residue Ser232, leads us to infer a direct contact between Lys529 and Glu233 for initiation of TP-DNA replication. Altogether, the results are compatible with a sequential binding of ?29 DNA polymerase residue Lys529 with TP and DNA during replication of TP-DNA.

SUBMITTER: del Prado A

PROVIDER: S-EPMC3762793 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.

del Prado Alicia A Lázaro José M JM Villar Laurentino L Salas Margarita M de Vega Miguel M

PloS one 20130904 9

Resolution of the crystallographic structure of φ29 DNA polymerase binary and ternary complexes showed that residue Lys529, located at the C-terminus of the palm subdomain, establishes contacts with the 3' terminal phosphodiester bond. In this paper, site-directed mutants at this Lys residue were used to analyse its functional importance for the synthetic activities of φ29 DNA polymerase, an enzyme that starts linear φ29 DNA replication using a terminal protein (TP) as primer. Our results show t ...[more]

PMID: 24023769

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Project description:BackgroundPolymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library.ResultsHere, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template.ConclusionsDNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially available DNA polymerases. The results suggest that the interaction of the DNA polymerase with the primer:template junction during the initiation of DNA polymerization is very important in terms of overall amplification bias and has broader implications for both the primer design process and multiplex PCR.

Dataset Information

Dual role of ?29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.

Publications

Dual role of φ29 DNA polymerase Lys529 in stabilisation of the DNA priming-terminus and the terminal protein-priming residue at the polymerisation site.

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