Visualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal

Kiefer, James R.; Chen, Mao; Braman, Jeffrey C.; Beese, L.S.

doi:10.1038/34693

Cited by 537 publications

(508 citation statements)

References 28 publications

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“…Both the ribozyme and protein enzymes suffer mere elemental effects upon substitution of the pro-S P oxygen and much larger effects with the pro-R P substitution. This leaves open the possibility that both the ribozyme and the protein active sites use the same strategy for promoting the chemistry of primer extension, each employing similar two-metal-ion mechanisms for phosphoryl transfer (21)(22)(23)(24). Furthermore, the similar elemental effects upon pro-S P substitution (37-and 60-fold) suggest that the two transition states lie at similar positions along the continuum between associative and dissociative character (15 (8,12).…”

Section: Resultsmentioning

confidence: 99%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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In support of the idea that certain RNA molecules might be able to catalyze RNA replication, a ribozyme was previously generated that synthesizes short segments of RNA in a reaction modeled after that of proteinaceous RNA polymerases. Here, we describe substrate recognition by this polymerase ribozyme. Altering base or sugar moieties of the nucleoside triphosphate only moderately affects its utilization, provided that the alterations do not disrupt Watson-Crick pairing to the template. Correctly paired nucleotides have both a lower K m and a higher k cat , suggesting that differential binding and orientation each play roles in discriminating matched from mismatched nucleotides. Binding of the pyrophosphate leaving group appears weak, as evidenced by a very inefficient pyrophosphate-exchange reaction, the reverse of the primer-extension reaction. Indeed, substitutions at the γ-phosphate can be tolerated, although poorly. Thio substitutions of oxygen atoms at the reactive phosphate exert effects similar to those seen with cellular polymerases, leaving open the possibility of an active site analogous to those of protein enzymes. The polymerase ribozyme, derived from an efficient RNA ligase ribozyme, can achieve the very fast k cat of the parent ribozyme when the substrate of the polymerase (GTP) is replaced by an extended substrate (pppGGA), in which the GA dinucleotide extension corresponds to the second and third nucleotides of the ligase. This suggests that the GA dinucleotide, which had been deleted when converting the ligase into a polymerase, plays an important role in orienting the 5′-terminal nucleoside. Polymerase constructs that restore this missing orientation function should achieve much more efficient and perhaps more accurate RNA polymerization.The RNA world hypothesis proposes that early life relied on catalytic RNAs rather than protein enzymes for catalysis (1). Much of the appeal of this theory comes from the notion that ribozymes, which could have served as their own genes, would have been much simpler to duplicate than proteins (2-5). Hence, a vital component of the RNA world scenario is an RNA polymerase ribozyme responsible for replicating the essential ribozymes of early life (including itself). Whether there might be RNA sequences that can promote RNA polymerization with sufficient accuracy and efficiency for RNA self-replication is one of the fundamental unanswered questions related to the RNA world hypothesis (1, 6). Finding such a sequence would support the idea of the RNA world and provide a key component for the synthesis of simple living cells (7).The class I ligase ( Figure 1A) is a promising starting point for efforts to develop a self-replicating ribozyme. This ribozyme ligates two RNAs, generating a 3′,5′-phosphodiester linkage and releasing pyrophosphate, a reaction similar to that of RNA and DNA polymerases (8). Engineered forms of the ligase can polymerize short segments of RNA ( Figure 1C). In the presence of appropriate RNA templates and nucleoside triphosphates (NTP...

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Section: Resultsmentioning

confidence: 99%

Recognition of Nucleoside Triphosphates during RNA-Catalyzed Primer Extension

et al. 2000

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“…Aside from translesion synthesis, it has recently been shown that at least some Y-family DNA polymerases are involved in other functions, including recombination and somatic hypermutation [8,9,66]. BF features a sterically tight active site and an exonuclease domain {non-functional in the strain used in crystallographic studies [11,31,62] but functional in Bst 320 ATCC55719 (http://www.patentstorm.us/patents/5747298-claims.html)}, but lacks a little finger domain, whereas Dpo4 lacks an exonuclease domain but has an additional little finger domain, and a sterically more open active site. Cartoon representations of BF (a) and Dpo4 (b).…”

Section: The High-fidelity Dna Polymerase Bfmentioning

confidence: 99%

“…Furthermore, in the active site region, fewer Dpo4 amino acid residues are in contact with the DNA:Dpo4 forms hydrogen bonds only with the phosphate groups of the DNA duplex region [13]. By contrast, BF forms additional extensive sequenceindependent interactions with the minor-groove edges of the first four base pairs at the growing end of the DNA duplex, with hydrogen bonds between the bases and highly conserved protein side chains (Box 4) or oriented water molecules anchored to protein side chains [31]. The sterically open Dpo4 active-site region is structured to enable the accommodation of DNA lesions, while facilitating low-fidelity DNA replication.…”

Section: The High-fidelity Dna Polymerase Bfmentioning

confidence: 99%

“…These interactions constitute a minor-groove scanning track or 'reading head' that enables the polymerase to sense an incorporated mismatch [31]; this proofreading promotes subsequent routing of the error-containing duplex for excision by an intrinsic exonuclease domain or an external exonuclease activity [10,59]. (Proofreading 3′→5′ exonuclease activity catalyzes the removal of mismatched nucleotides from the growing end of the primer strand.)…”

Section: Box 4 Key Structural Differences Between Bf and Dpo4mentioning

confidence: 99%

“…(Proofreading 3′→5′ exonuclease activity catalyzes the removal of mismatched nucleotides from the growing end of the primer strand.) As part of the minor-groove scanning track, two strictly conserved 'reading' residues, Arg615 and Gln797 in the case of BF, form hydrogen bonds with the newly formed base pair, ensuring its proper geometry [31]. Gln797 interacts with the template base and Arg615 interacts with the dNTP.…”

Section: Box 4 Key Structural Differences Between Bf and Dpo4mentioning

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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