Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution

Kiefer, James R.; Chen, Mao; Hansen, Connie; Basehore, Scott L; Hogrefe, Holly H.; Braman, Jeffrey C.; Beese, L.S.

doi:10.1016/s0969-2126(97)00169-x

Cited by 151 publications

(157 citation statements)

References 66 publications

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“…Wild-type and D598A/ F710Y mutant proteins were purified as described (13). The D598A/F710Y double mutant (34) was used to capture ternary complexes.…”

Section: Methodsmentioning

confidence: 99%

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

Wang

Hellinga²,

Beese³

2011

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

235

276

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Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C•A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.crystal structure | replication fidelity | mispair | polymerase structure H igh-fidelity polymerases replicate double-stranded DNA with remarkable accuracy (1). Fidelity is achieved by a successive series of conformational changes and molecular recognition events encoded at different sites on the polymerase surface such that mismatches are either prevented from incorporating or are excised within a few nucleotides past their incorporation point (2-5). At the site of covalent incorporation, shape complementarity between the polymerase surface and the edges of correctly paired bases is the dominant mechanism that determines specificity (6, 7). Here, mismatched base pairs or lesions that do not conform to this stereochemical constraint misalign their incoming triphosphate moiety relative to the 3′ OH of the growing primer terminus, leading to rejection of the incorrect or damaged nucleotides (2-4). However, modified bases that maintain the stereochemistry of cognate base-pair edges are readily incorporated (6-8). Nevertheless, polymerases do incorporate mismatched nucleotide base pairs at low frequency, leading to spontaneous mutagenesis (1).The mechanism by which spontaneous replication errors occur has long been the subject of intense speculation. In their second paper on the structure of DNA, Watson and Crick recognized that tautomerization alters the hydrogen-bonding patterns and therefore could enable mismatches to assume the structure of canonical base pairs (9). This notion was elaborated in the rare tautomer hypothesis of spontaneous mutagenesis, which states that mutations arise through the formation of high-energy tautomers at low frequency (8, 10). However, it has been challenging to obtain direct structural evidence for this mechanism. In the absence of polymerase, mismatches do not adopt a canonical base-pair structure in DNA (5, 11). Recently, a T•G mismatch has been observed to adopt a canonical base-pair structure in a polymerase, due to an ionization event, demonstrating that noncanonical hydrogen-bonding patt...

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“…Wild-type and D598A/ F710Y mutant proteins were purified as described (13). The D598A/F710Y double mutant (34) was used to capture ternary complexes.…”

Section: Methodsmentioning

confidence: 99%

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

Wang

Hellinga²,

Beese³

2011

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

235

276

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“…Wild-type and mutant proteins were purified as described in ref. 42. Wild-type protein was used for all kinetics experiments and all crystallizations except ddTTP/ddCT-P:BF:DNA ternary complexes, which were grown by using BF D598/F710Y.…”

Section: Methodsmentioning

confidence: 99%

The structural basis for the mutagenicity of O ⁶ -methyl-guanine lesions

Warren

Forsberg

Beese

2006

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

133

178

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Methylating agents are widespread environmental carcinogens that generate a broad spectrum of DNA damage. Methylation at the guanine O 6 position confers the greatest mutagenic and carcinogenic potential. DNA polymerases insert cytosine and thymine with similar efficiency opposite O 6 -methyl-guanine (O6MeG). We combined pre-steady-state kinetic analysis and a series of nine x-ray crystal structures to contrast the reaction pathways of accurate and mutagenic replication of O6MeG in a high-fidelity DNA polymerase from Bacillus stearothermophilus. Polymerases achieve substrate specificity by selecting for nucleotides with shape and hydrogen-bonding patterns that complement a canonical DNA template. Our structures reveal that both thymine and cytosine O6MeG base pairs evade proofreading by mimicking the essential molecular features of canonical substrates. The steric mimicry depends on stabilization of a rare cytosine tautomer in C⅐O6MeG-polymerase complexes. An unusual electrostatic interaction between O-methyl protons and a thymine carbonyl oxygen helps stabilize T⅐O6MeG pairs bound to DNA polymerase. Because DNA methylators constitute an important class of chemotherapeutic agents, the molecular mechanisms of replication of these DNA lesions are important for our understanding of both the genesis and treatment of cancer.crystal structure ͉ DNA damage ͉ DNA polymerase ͉ protein-DNA complex A lkylating agents are potent environmental carcinogens that are produced by burning tobacco or in grilling foods (1, 2) and also may be formed enzymatically in vivo (3, 4), for instance, by enzymatic metabolite nitrosation (5). Such agents cause a broad spectrum of DNA lesions. Although modifications at the O6 position of guanine constitute a minority of the total lesions, they are the most carcinogenic (6-8). The cytotoxic effects of DNA-methylating agents have been exploited in their use as potent anticancer agents. O 6 -methyl-guanine (O6MeG) is mutagenic because polymerases frequently misinsert T opposite O6MeG instead of C, both in vivo (9, 10) and in vitro (11)(12)(13). In this study, we present the crystal structures of complexes of a high-fidelity DNA polymerase with substrates representing several steps of nucleotide insertion opposite O6MeG. Additionally, we have engineered a substrate in which the O6MeG⅐C/T pair lies in DNA outside the binding site of the polymerase, allowing us to compare the conformation of these base pairs in duplex DNA to the conformation of the base pairs constrained in the polymerase active site.The relative preference for incorporation of T and C opposite an O6MeG lesion varies somewhat with polymerase and sequence context (13). High-fidelity polymerases such as exonuclease-deficient E. coli polymerase I (Klenow fragment) (13) or bacteriophage T7 DNA polymerase (11) show an Ϸ7-fold preference for misinsertion of T. By comparison, these polymerases usually show a several thousand-fold preference for insertion of a correct base-pairing partner when copying a normal, undamaged DNA. O6MeG lesions ...

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“…Specifically, the Y-family polymerase Dpo4 has a mismatch error rate of ~6.5 × 10 −3 and a single-base-deletion error rate of ~2.4 × 10 −3 [60], whereas the A-family polymerase BF has an error rate of ~1.5 × 10 −5 (http://www.freepatentsonline.com/5834253.html). Polymerase processivity, namely the number of nucleotides added per binding event, is only one or two nucleotides for Dpo4 without additional cofactors in vitro [61], whereas, for BF, it is 111 nucleotides [62]. The presence of accessory proteins in the holoenzyme in vivo increases DNA-synthesis processivity thousands of folds for high-fidelity polymerases, but only slightly for lesionbypass DNA polymerases [63,64].…”

Section: The High-fidelity Dna Polymerase Bfmentioning

confidence: 99%

“…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%

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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Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution

Cited by 151 publications

References 66 publications

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

The structural basis for the mutagenicity of O ⁶ -methyl-guanine lesions

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

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Crystal structure of a thermostable Bacillus DNA polymerase l large fragment at 2.1 Å resolution

Cited by 151 publications

References 66 publications

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

The structural basis for the mutagenicity of O 6 -methyl-guanine lesions

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

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The structural basis for the mutagenicity of O ⁶ -methyl-guanine lesions