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Closed Access item A Conserved Insertion in Protein-primed DNA Polymerases is Involved in Primer Terminus Stabilisation

Authors:Dufour, Emmanuelle
Rodríguez, Irene
Lázaro, José M.
Vega, Miguel de
Salas, Margarita
Keywords:Linear DNA replication, Protein priming, Bacteriophage φ29
Issue Date:22-Aug-2003
Publisher:Elsevier
Citation:Journal of Molecular Biology 331(4): 781-794 (2003)
Abstract:Protein-primed DNA polymerases form a subgroup of the eukaryotic-type DNA polymerases family, also called family B or α-like. A multiple amino acid sequence alignment of this subgroup of DNA polymerases led to the identification of two insertions, TPR-1 and TPR-2, in the polymerisation domain. We showed previously that Asp332 of the TPR-1 insertion of ø29 DNA polymerase is involved in the correct orientation of the terminal protein (TP) for the initiation of replication. In this work, the functional role of two other conserved residues from TPR-1, Lys305 and Tyr315, has been analysed. The four mutant derivatives constructed, K305I, K305R, Y315A and Y315F, displayed a wild-type 3′–5′ exonuclease activity on single-stranded DNA. However, when assayed on double-stranded DNA such activity was higher than that of the wild-type enzyme. This activity led to a reduced pol/exo ratio, suggesting a defect in stabilising the primer terminus at the polymerase active site. On the other hand, although mutant polymerases K305I and Y315A were able to couple processive DNA polymerisation to strand displacement, they were severely impaired in ø29 TP-DNA replication. The possible role of the TPR-1 insertion in the set of interactions with the nascent chain during the first steps of TP-DNA replication is discussed.
Publisher version (URL):http://dx.doi.org/10.1016/S0022-2836(03)00788-5
URI:http://hdl.handle.net/10261/38995
ISSN:0022-2836
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Appears in Collections:(CBM) Artículos

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