Crystal Structures of Human DNA Polymerase β Complexed with DNA:  Implications for Catalytic Mechanism, Processivity, and Fidelity<sup>,</sup>

Pelletier, H.; Sawaya, M.R.; Wolfle, William T.; Wilson, Samuel H.; Kraut, Joseph

doi:10.1021/bi952955d

Cited by 273 publications

(291 citation statements)

References 78 publications

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“…6; strand 5 also includes the catalytic residue Asp-256 (not shown). The loop itself is disordered or solventexposed in crystal structures and is positioned at some distance from the active site (30,31). This makes it difficult to deduce what the role of this loop in substrate interactions could be.…”

Section: More Efficient Mispair Extension May Underlie the Mutatormentioning

confidence: 99%

The E249K Mutator Mutant of DNA Polymerase β Extends Mispaired Termini

Kosa

Sweasy

1999

Journal of Biological Chemistry

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The DNA polymerase ␤ mutant enzyme, which is altered from glutamic acid to lysine at position 249, exhibits a mutator phenotype in primer extension assays and in the herpes simplex virus-thymidine kinase (HSV-tk) forward mutation assay. The basis for this loss of accuracy was investigated by measurement of misincorporation fidelity in single turnover conditions. For the four misincorporation reactions investigated, the fidelity of the E249K mutant was not significantly different from wild type, implying that the mutator phenotype was not caused by a general inability to distinguish between correct and incorrect bases during the incorporation reaction. However, the discrimination between correct and incorrect substrates by the E249K enzyme occurred less during the conformational change and chemical steps and more during the initial binding step, compared with pol ␤ wild type. This implies that the E249K mutation alters the kinetic mechanism of nucleotide discrimination without reducing misincorporation fidelity. In a missing base primer extension assay, we observed that the mutant enzyme produced mispairs and extended them. This indicates that the altered fidelity of E249K could be due to loss of discrimination against mispaired primer termini. This was supported by the finding that the E249K enzyme extended a G:A mispair 8-fold more efficiently than wild type and a C:T mispair 4-fold more efficiently. These results demonstrate that an enhanced ability to extend mispairs can produce a mutator phenotype and that the Glu-249 side chain of DNA polymerase ␤ is critical for mispair extension fidelity. DNA polymerase ␤ (pol ␤)1 is a mammalian polymerase that fills short gaps in DNA (1, 2). pol ␤ has been implicated in base excision repair (3, 4) and appears to function during meiosis (5).Homozygous deletion of the pol ␤ gene in mice is an embryonic lethal event (6), indicating that pol ␤ activity is absolutely required during development, although its specific role is not clear. The best characterized function of pol ␤ is gap-filling synthesis during base excision repair (BER). The BER pathway repairs oxidative and alkylation damage, as well as other DNA lesions. After excision of a damaged base by a DNA glycosylase, AP endonuclease (APE) binds to the abasic site and recruits pol ␤ into a complex with the APE and DNA (7). APE incises the DNA, and pol ␤ conducts both the gap-filling synthesis and the deoxyribophosphatase steps (1,8,9). As AP sites are believed to occur in eukaryotic cells about 10,000 times per cell per day (10), this repair pathway is vital for genomic stability.pol ␤ has also been implicated in the process of meiotic recombination. Plug et al. (5) found that pol ␤ dynamically localizes to the synaptonemal complexes formed by chromosome pairs during meiosis. For this reason, pol ␤ is suspected of participating in meiosis.pol ␤ may also have a role in DNA replication. It is required for conversion of single-stranded to double-stranded DNA in Xenopus extracts (11) and is able to join Okazaki fragments (...

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Section: More Efficient Mispair Extension May Underlie the Mutatormentioning

confidence: 99%

The E249K Mutator Mutant of DNA Polymerase β Extends Mispaired Termini

Kosa

Sweasy

1999

Journal of Biological Chemistry

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“…Isoleucine 260 is located within a hydrophobic hinge region that appears to function in the movement of the fingers subdomain upon interaction of the polymerase with its nucleotide substrate (14). Alteration of other hinge residues of pol ␤ results in polymerases with strong mutator activity, both in vivo and in vitro (15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

“…Lysine 289 is located in ␣-helix N of the pol ␤ protein (14), which is important for accurate DNA synthesis (20)(21)(22)(23) because of its role in positioning the templating base during phosphodiester bond formation. We previously showed that the K289M protein (24) induces mutations within interrupted runs of like nucleotides in mouse cells at a 16-fold frequency over that of the WT enzyme.…”

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confidence: 99%

Expression of DNA polymerase β cancer-associated variants in mouse cells results in cellular transformation

Sweasy

Lang

Starcevic

et al. 2005

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

110

144

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Thirty percent of the 189 tumors studied to date express DNA polymerase ␤ variants. One of these variants was identified in a prostate carcinoma and is altered from isoleucine to methionine at position 260, within the hydrophobic hinge region of the protein.Another variant was identified in a colon carcinoma and is altered at position 289 from lysine to methionine, within helix N of the protein. We have shown that the types of mutations induced by these cancer-associated variants are different from those induced by the wild-type enzyme. In this study, we show that expression of the I260M and K289M cancer-associated variants in mouse C127 cells results in a transformed phenotype in the great majority of cell clones tested, as assessed by focus formation and anchorageindependent growth. Strikingly, cellular transformation occurs after a variable number of passages in culture but, once established, does not require continuous expression of the polymerase ␤ variant proteins, implying that it has a mutational basis. Because DNA polymerase ␤ functions in base excision repair, our results suggest that mutations that arise during this process can lead to the onset or progression of cancer.base excision repair ͉ DNA repair ͉ mutagenesis S pontaneous DNA damage occurs at a rate of Ϸ10,000 lesions per cell per day, and much of this damage is repaired by the base excision repair (BER) machinery (1, 2). The BER system plays a critical role in maintaining cellular genomic stability. During BER, damaged bases are removed by a DNA glycosylase, followed by incision of the DNA by AP endonuclease (APE) at a position that is usually 5Ј to the lesion, leaving a nick with a 3ЈOH and a 5Ј deoxyribose phosphate (2). DNA polymerase beta (pol ␤) binds to the nick, removes the deoxyribose phosphate with its DRP lyase activity (3), and fills in the single nucleotide gap, using its DNA polymerase activity (4).Fifty-eight of the 189 tumors characterized to date express DNA pol ␤ variant proteins (for review, see ref. 5) (6). Of these, 28 (48%) expressed variants with single amino acid alterations, seven expressed truncated forms of pol ␤, and eight expressed multiple variant forms of pol ␤. These mutations are absent from normal tissue from the same individuals and are not among the common polymorphisms found within the pol ␤ gene (http:͞͞ egp.gs.washington.edu͞data͞polb) (5, 7). In addition, an alternative splice variant of pol ␤ in which exon 11 is deleted was expressed in 15 tumors. This splice variant appears to interfere with BER (8). This exon 11 splice variant has been detected in normal tissue, including normal tissue isolated from 2 of 15 patients with tumors, and its link to cancer etiology remains controversial (9-12). Each of the tumors characterized to date also contain the wild-type (WT) pol ␤ allele.The I260M variant of pol ␤ was identified in a prostate carcinoma (13). Isoleucine 260 is located within a hydrophobic hinge region that appears to function in the movement of the fingers subdomain upon interaction of the polymeras...

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“…Pol ␤ lacks an intrinsic proofreading exonuclease and generates singlebase substitution errors at rates of 0.5-13 ϫ 10 Ϫ4 when filling short gaps (16,17). This fidelity is higher than predicted by free energy differences between correct and incorrect base pairing in solution (18), suggesting that like other DNA polymerases, pol ␤ contributes to the selectivity of nucleotide incorporation.Structural and biochemical evidence suggests that nucleotide selection by pol ␤ occurs at a step following initial, nonspecific dNTP binding (5,6,19,20). When DNA with a singlenucleotide gap is bound by pol ␤, a 90°kink is observed at the 5Ј-phosphodiester linkage of the template residue that base pairs with the incoming dNTP (5, 6).…”

mentioning

confidence: 99%

“…Structural and biochemical evidence suggests that nucleotide selection by pol ␤ occurs at a step following initial, nonspecific dNTP binding (5,6,19,20). When DNA with a singlenucleotide gap is bound by pol ␤, a 90°kink is observed at the 5Ј-phosphodiester linkage of the template residue that base pairs with the incoming dNTP (5, 6).…”

mentioning

confidence: 99%

Base Substitution Specificity of DNA Polymerase β Depends on Interactions in the DNA Minor Groove

Osheroff¹,

Beard²,

Wilson³

et al. 1999

Journal of Biological Chemistry

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To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase ␤ mutants at residue Arg 283, where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T⅐dGMP and A⅐dGMP mispairs but reduced selectivity for T⅐dCMP and A⅐dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.Accurate DNA synthesis catalyzed by DNA polymerases during repair and replication is essential for genome stability. Thus, it is important to understand how DNA polymerases select correct nucleotides for incorporation. That polymerases may distinguish correct from incorrect base pairs by examining the positions of hydrogen bonding atoms in the minor groove of the template⅐primer duplex was suggested more than 20 years ago (1, 2). The positions of the two minor groove hydrogen bond acceptors (N3 of purines and O2 of pyrimidines) are similar in Watson-Crick base pairs, but different in mismatched base pairs (3, 4). The structure of a DNA polymerase ␤⅐DNA⅐ddNTP complex reveals that the active site binding pocket for the nascent base pair is partly formed by interactions of side chains with the DNA minor groove (5, 6). Structural studies of Pol I family polymerases from Thermus aquaticus (7,8), Escherichia coli (9), bacteriophage T7 (10), and Bacillus stearothermophilus (11), and a Pol ␣ family polymerase from bacteriophage RB69 (12) reveal numerous interactions (hydrogen bonds and van der Waals contacts) in the DNA minor groove, both at and upstream of the active site.Here we probe the importance of polymerase interactions with the DNA minor groove using DNA pol 1 ␤, the smallest and most extensively studied mammalian DNA polymerase. Pol ␤'s primary function is in base excision repair (BER) of DNA damage resulting from exposure to endogenous metabolites and reactive genotoxicants (13)(14)(15). In this capacity, pol ␤ fills in gaps of a single nucleotide (single-nucleotide BER) or gaps of two to six nucleotides (alternate or long patch BER). Pol ␤ lacks an intrinsic proofreading exonuclease and generates singlebase substitution errors at rates of 0.5-13 ϫ 10 Ϫ4 when filling short gaps (16,17). This fidelity is higher than predicted by free energy differences between correct and incorrect base pairing in solution (18), suggesting th...

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Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity^,

Cited by 273 publications

References 78 publications

The E249K Mutator Mutant of DNA Polymerase β Extends Mispaired Termini

The E249K Mutator Mutant of DNA Polymerase β Extends Mispaired Termini

Expression of DNA polymerase β cancer-associated variants in mouse cells results in cellular transformation

Base Substitution Specificity of DNA Polymerase β Depends on Interactions in the DNA Minor Groove

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Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity,

Cited by 273 publications

References 78 publications

The E249K Mutator Mutant of DNA Polymerase β Extends Mispaired Termini

The E249K Mutator Mutant of DNA Polymerase β Extends Mispaired Termini

Expression of DNA polymerase β cancer-associated variants in mouse cells results in cellular transformation

Base Substitution Specificity of DNA Polymerase β Depends on Interactions in the DNA Minor Groove

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Product

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Crystal Structures of Human DNA Polymerase β Complexed with DNA: Implications for Catalytic Mechanism, Processivity, and Fidelity^,