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

Dufour, Emmanuelle; Rodrı́guez, Irene; Lázaro, José Portolés; Vega, Miguel de; Salas, Margarita

doi:10.1016/s0022-2836(03)00788-5

Cited by 17 publications

(16 citation statements)

References 54 publications

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“…29 DNA polymerase possesses two insertions in the palm subdomain, specifically conserved in the subgroup of DNA polymerases that use a protein as a primer (26). They are TPR1, whose conserved residues were proposed to make contacts with the TP and DNA (25,27), and TPR2 with a biochemically uncharacterized functional role (see Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

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A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

Rodrı́guez

Lázaro

Blanco

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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101

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Recent crystallographic studies of 29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a 29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of 29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.protein-primed replication ͉ terminal protein region ͉ helicase-like activity ͉ DNA-binding stability D NA replication is a complex multistep process that involves a wide range of proteins and enzymatic activities (1, 2). DNA synthetic activity is provided by DNA polymerases that add nucleotides to the 3Ј-OH end of a primer strand guided by base pairing with the template strand. Polymerases involved in DNA replication are referred to as replicases to distinguish them from other DNA polymerases whose synthetic activities play a role in processes such as DNA repair or recombination. In most DNA replication systems, replication fork movement along the duplex DNA requires an unwinding activity to separate the strands as replication progresses (1, 2). Generally, such activity is not intrinsic to the replicase but is provided either by monomeric or multimeric enzymes called helicases, which melt the dsDNA in an ATP-dependent fashion. In addition, the intrinsic processivity (number of nucleotides incorporated per single DNA polymerase͞DNA-binding event) of most replicases is not high enough to account for the replication of an entire genome, and therefore processivity factors are also required to hold the DNA replicase on the template strand (1, 2).Bacteriophage 29 DNA polymerase is a protein-primed DNA-dependent replicase belonging to the eukaryotic-type family of DNA polymerases (family B). Other members of this family include polymerases with cellular, bacterial, and viral origins (3). 29 DNA polymerase, like many other replicative polymerases, contains both 5Ј-3Ј synthetic and 3Ј-5Ј degradative activities within a single polypeptide chain. Its intrinsic insertion discrimination of 10 4 to 10 6 (4) is further improved 100-fold (5) through proofreading by the exonuclease domain. An extensive mutational analysis of 29 DNA polymerase served to identify the catalytic residues required for these two activities, as well as those responsible for the stabilization of the primer terminus at the respective active sites; these residues are evolutionarily conserved in most DNA polymerases (reviewed ...

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Section: Resultsmentioning

confidence: 99%

“…25 and 26. Mutational analysis of TPR1 indicated its involvement in interactions with both TP and DNA substrates (25,27). Although mutational data on TPR2 were unavailable, homology modeling of the DNA from the RB69 DNA polymerase ternary complex (28) onto the structure of 29 DNA polymerase suggested possible functional roles.…”

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confidence: 99%

A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

Rodrı́guez

Lázaro

Blanco

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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101

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“…Partial proteolysis of the f29 DNA polymerase/TP heterodimer resulted in the same protection of the central cleavage site as obtained upon DNA binding, suggesting that a similar conformational change occurs upon TP binding and that both primers, DNA and TP, fit in the same dsDNA binding channel (de Vega et al, 1998;Truniger et al, 2000). In agreement with this, most f29 DNA polymerase residues involved in DNA binding are also important for TP binding (Méndez et al, 1994;Blasco et al, 1995;de Vega et al, 1998;Truniger et al, 1996Truniger et al, , 1998Truniger et al, , 1999Eisenbrandt et al, 2002;Rodríguez et al, 2003;Dufour et al, 2000Dufour et al, , 2003. Of the mutant DNA polymerases studied here the three with greater impairment to start replication, K196I, F198V and K206I, showed reduced TPdAMP formation activities in the absence of TP-DNA.…”

Section: Discussionmentioning

confidence: 74%

Involvement of the “linker” region between the exonuclease and polymerization domains of ϕ29 DNA polymerase in DNA and TP binding

Truniger

Bonnin

Lázaro

et al. 2005

Gene

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“…A region reminiscent of the so-called TPR- 1B). In phage 29 DNA polymerase, TPR-1 was demonstrated to be important for positioning of the protein primer and for transition between protein-primed and DNA-primed modes of replication (14,15,27,37). A sequence corresponding to motif C of DNA polymerases (YX DTDS), which via its Asp residues contributes to metal ion binding and catalysis (7), was not obvious in REP pAL1 .…”

Section: Resultsmentioning

confidence: 99%

“…The consensus sequence of motifs A and B of 29 DNA polymerase, which is conserved among different nucleic-acid-synthesizing enzymes (7), is indicated above the sequences. Residues shaded in gray that are marked with a diamond above the sequence may represent conserved residues of the TPR-1 region, which is specific to protein-priming DNA polymerases (14,15,27,37). (C) Alignment of a region rich in acidic residues, which is highly conserved among REP pAL1 (aa 1635 to 1702) and rhodococcal homologs.…”

Section: Resultsmentioning

confidence: 99%

A Novel Replicative Enzyme Encoded by the LinearArthrobacterPlasmid pAL1

Kolkenbrock

Naumann²,

Hippler³

et al. 2010

J Bacteriol

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The soil bacterium Arthrobacter nitroguajacolicus Rü61a contains the linear plasmid pAL1, which codes for the degradation of 2-methylquinoline. Like other linear replicons of actinomycetes, pAL1 is characterized by short terminal inverted-repeat sequences and terminal proteins (TP pAL1 ) covalently attached to its 5 ends. TP pAL1 , encoded by the pAL1.102 gene, interacts in vivo with the protein encoded by pAL1.101. Bioinformatic analysis of the pAL1.101 protein, which comprises 1,707 amino acids, suggested putative zinc finger and topoisomerase-primase domains and part of a superfamily 2 helicase domain in its N-terminal and central regions, respectively. Sequence motifs characteristic of the polymerization domain of family B DNA polymerases are partially conserved in a C-terminal segment. The purified recombinant protein catalyzed the deoxycytidylation of TP pAL1 in the presence of single-stranded DNA templates comprising the 3-terminal sequence (5-GCAGG-3), which in pAL1 forms the terminal inverted repeat, but also at templates with 5-(G/T)CA(GG/GC/CG)-3 ends. Enzyme assays suggested that the protein exhibits DNA topoisomerase, DNA helicase, and DNA-and protein-primed DNA polymerase activities. The pAL1.101 protein, therefore, may act as a replicase of pAL1.Linear plasmids have been identified in higher plants, fungi, and many bacteria. Most of these linear genetic elements are characterized by terminal inverted-repeat sequences and terminal proteins (TPs) covalently attached to their 5Ј ends. The presence of TPs is a consequence of their mode of DNA replication, which in linear plasmids of plants, yeasts, and fungi is initiated at the termini by using the TP as a primer and proceeds by strand displacement. The DNA polymerases encoded by these linear elements are of the viral B type, related to those of contemporary adenoviruses and Bacillus phages (29). The mechanism of protein-primed DNA replication has been studied in detail, especially for the model of Bacillus subtilis phage 29, which uses a monomeric B-family DNA polymerase for both the TP-primed initiation reaction and DNA elongation, resulting in continuous, full-length replication of both strands of the 29 genome (5,6,7,26,42,43,44). In contrast to the linear plasmids of eukaryotes, linear chromosomes and plasmids of Streptomyces spp. replicate bidirectionally from an internal origin (9). This replication mechanism encounters the problem that discontinuous lagging-strand synthesis from RNA-primed Okazaki fragments leaves recessed 5Ј ends at both telomeres when the distal RNA primers are removed. In the Streptomyces linear replicons, the TP serves as a primer for filling in these single-stranded gaps (2, 58). Both the TP and a telomere-associated protein (Tap), which is presumed to recruit the TP and position it at the telomere, are necessary for the propagation of Streptomyces replicons in their linear forms (3).The TP and the Tap protein are highly conserved among many Streptomyces species. On the other hand, several studies have suggested a conside...

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

Cited by 17 publications

References 54 publications

A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity

Involvement of the “linker” region between the exonuclease and polymerization domains of ϕ29 DNA polymerase in DNA and TP binding

A Novel Replicative Enzyme Encoded by the LinearArthrobacterPlasmid pAL1

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