Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Berman, Andrea J.; Kamtekar, S.; Goodman, Jessica L.; Lázaro, José Portolés; Vega, Miguel de; Blanco, Luis; Salas, Margarita; Steitz, Thomas A.

doi:10.1038/sj.emboj.7601780

Cited by 142 publications

(253 citation statements)

References 54 publications

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“…Translocation is the processive movement of a polymerase relative to the DNA duplex between two rounds of the phosphoryl-transfer reaction during polynucleotide replication [32]; it is essential for polymerase function and necessarily occurs with every polymerase upon completion of one replication cycle ( Figure 1). Although the actual dynamic mechanism of translocation is not known for any polymerase, static crystal structures can indicate plausible intermediate states in this process.…”

Section: Induced Fit and Translocation: Differences Between Bf And Dpo4mentioning

confidence: 99%

“…By contrast, the BF crystal structures indicate that the entire translocation cycle occurs following dNTP entry into the BF active site and the induced-fit polymerase closing. The driving forces that could power translocation have been discussed based on crystal structures of a B-family DNA polymerase, phi29 from Bacillus subtilis [32]: dissociation of pyrophosphate breaks the electrostatic link between the fingers domain and the catalytic Mg 2+ ions, enabling the fingers to pivot to the open position. Then, two conserved tyrosine residues enter the insertion site and sterically exclude the nascent base pair.…”

Section: Dpo4: Stepwise Translocationmentioning

confidence: 99%

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Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

Broyde¹,

Wang²,

Rechkoblit³

et al. 2008

Trends in Biochemical Sciences

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When a high-fidelity DNA polymerase encounters certain DNA-damage sites, its progress can be stalled and one or more lesion-bypass polymerases are recruited to transit the lesion. Here, we consider two representative types of lesions: (i) 7,8-dihydro-8-oxogua-nine (8-oxoG), a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo [a]pyrene, in the high-fidelity DNA polymerase Bacillus fragment (BF) from Bacillus stearothermophilus and in the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. The tight fit of the BF polymerase around the nascent base pair contrasts with the more spacious, solvent-exposed active site of Dpo4, and these differences in architecture result in distinctions in their respective functions: one-step versus stepwise polymerase translocation, mutagenic versus accurate bypass of 8-oxoG, and polymerase stalling versus mutagenic bypass at bulky benzo[a]pyrene-derived lesions. Lesions and DNA polymerasesUntil 1999, it was widely understood that bacteria have three DNA polymerases and mammals have five [1]. In 1999, however, an entirely new category of polymerases was discovered in prokaryotes and eukaryotes [2-4] -the lesion-bypass DNA polymerases. The function of lesion-bypass polymerases is to replicate past lesion-containing DNA templates in a process called translesion synthesis [5][6][7]. Now at least 16 DNA polymerases are known in eukaryotes [8] and five in bacteria [9]. Furthermore, since 1999, crystal structures of DNA polymerases, including those containing lesions, have provided new structural insights into the mechanistic aspects of translesion synthesis and polymerase stalling associated with DNA lesions. For a review of the structures and mechanisms of DNA polymerases up to the year 2005, see Ref.[10].Here, we discuss the structural and functional features of representative high-fidelity and lesion-bypass DNA polymerases (Box 1) and how these features affect the processing of certain forms of DNA damage. The lesions considered are derived from reactions of intermediates produced by endogenous sources or from the metabolic activation of environmental genotoxic

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Section: Induced Fit and Translocation: Differences Between Bf And Dpo4mentioning

confidence: 99%

Section: Dpo4: Stepwise Translocationmentioning

confidence: 99%

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

Broyde¹,

Wang²,

Rechkoblit³

et al. 2008

Trends in Biochemical Sciences

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“…This mismatch-induced stalling, often referred to as "short-term memory," is a consequence of long-range distortions in the DNA that affect the conformation of the protein active site, mainly by template strand-mediated distortions (51). In positions −3 and −4, the template base moves into the minor groove in a conformation that is stabilized by direct contacts between the enzyme and the DNA backbone and solvent-mediated contacts in the minor groove; however, minor groove interactions on Φ29DNAP: DNA complex are stabilized by conserved residues in the thumb subdomain (52,53), thanks to interactions with the primer strand bases or sugar-phosphate backbone up to the −5 position (52). Similarly, the thumb subdomain of B35DNAP would provide interactions with the mismatched wobble up to one helix turn, providing a "memory" during stepwise polymerization.…”

Section: B35dnap Tls Is Counteracted By Proofreading Activity Up To 5 Ntmentioning

confidence: 99%

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

Berjón-Otero

Villar

Vega

et al. 2015

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

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DNA polymerases (DNAPs) responsible for genome replication are highly faithful enzymes that nonetheless cannot deal with damaged DNA. In contrast, translesion synthesis (TLS) DNAPs are suitable for replicating modified template bases, although resulting in very lowfidelity products. Here we report the biochemical characterization of the temperate bacteriophage Bam35 DNA polymerase (B35DNAP), which belongs to the protein-primed subgroup of family B DNAPs, along with phage Φ29 and other viral and mobile element polymerases. B35DNAP is a highly faithful DNAP that can couple strand displacement to processive DNA synthesis. These properties allow it to perform multiple displacement amplification of plasmid DNA with a very low error rate. Despite its fidelity and proofreading activity, B35DNAP was able to successfully perform abasic site TLS without template realignment and inserting preferably an A opposite the abasic site (A rule). Moreover, deletion of the TPR2 subdomain, required for processivity, impaired primer extension beyond the abasic site. Taken together, these findings suggest that B35DNAP may perform faithful and processive genome replication in vivo and, when required, TLS of abasic sites.protein-primed DNA polymerase | Bam35 | abasic sites | translesion synthesis | isothermal DNA amplification R eplicative DNA polymerases (DNAPs) from A and B families, collectively termed replicases, exhibit a "tight fit" for their DNA and dNTP substrates and are wondrously adapted to form correct Watson-Crick base pairs, resulting in very pronounced fidelity (1, 2). This strict preference to produce A:T and G:C base pairs is also the Achilles heel of faithful DNA polymerases, however, because they are strongly inhibited by modified nucleotides present at sites of DNA damage, leading to the stalling of replication fork and eventually to replicative stress and cell death (3). At the stalled replication fork, the DNA polymerase may be exchanged by a translesion synthesis (TLS) polymerase, generally belonging to the Y family. These enzymes possess looser solventexposed active sites, which allows them to deal with aberrant DNA features much better, although with a very low polymerization accuracy with the risk of the accumulation of mutations and genetic instability (4, 5). Alternatively, nonbulky modified bases, such as uracil and 8-oxo-deoxyguanosine (8oxoG), can be bypassed by replicases, with faithful or mutagenic outcomes that can be modified by the sequence context and dNTP availability (6, 7).Abasic or apurinic/apyrimidinic (AP) sites are the most common DNA lesions arising in cells when the N-glycosydic bond between the sugar moiety and the nucleobase is broken, either spontaneously or by a DNA glycosylase reaction product in the base excision repair pathway (8, 9). Unrepaired abasic sites are highly blocking lesions for replicative DNA polymerases (10), although mutant polymerases with impaired proofreading activity or with mutations in the polymerization active site residues that affect the incoming nucleotide sel...

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“…We use φ29, a polymerase known for its exceptional strand displacement activity, to push a DNA cargo. Researchers have studied the structure of φ29 polymerase and have provided useful insights into its exceptional strand displacement and processivity, and have deduced its translocation mechanism [9,16,17,32].…”

Section: Our Contributionmentioning

confidence: 99%

A DNA Nanotransport Device Powered by Polymerase ϕ29

2008

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Abstract. Polymerases are a family of enzymes responsible for copying or replication of nucleic acids (DNA or RNA) templates and hence sustenance of life processes. In this paper, we present a method to exploit a strand-displacing polymerase φ29 as a driving force for nanoscale transportation devices. The principle idea behind the device is strong strand displacement ability of φ29, which can displace any DNA strand from its template while extending a primer hybridized to the template. This capability of φ29 is used to power the movement of a target nanostructure on a DNA track. The major advantage of using a polymerase driven nanotransportation device as compared to other existing nanorobotical devices is its speed. φ29 polymerase can travel at the rate of 2000 nucleotides per minute [1] at room temperature, which translates to approximately 680 nanometers per minute on a nanostructure. We also demonstrate transportation of a DNA cargo on a DNA track with the help of fluorescence resonance electron transfer (FRET) data.

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Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Cited by 142 publications

References 54 publications

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

A DNA Nanotransport Device Powered by Polymerase ϕ29

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