共查询到20条相似文献,搜索用时 15 毫秒
1.
Tomas T. Richardson Xiaohua Wu Brian J. Keith Pauline Heslop Anita C. Jones Bernard A. Connolly 《Nucleic acids research》2013,41(4):2466-2478
Archaeal family-B DNA polymerases bind tightly to deaminated bases and stall replication on encountering uracil in template strands, four bases ahead of the primer-template junction. Should the polymerase progress further towards the uracil, for example, to position uracil only two bases in front of the junction, 3′–5′ proof-reading exonuclease activity becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and permanent mutation in 50% of the progeny. This publication uses both steady-state and time-resolved 2-aminopurine fluorescence to show pronounced unwinding of primer-templates with Pyrococcus furiosus (Pfu) polymerase–DNA complexes containing uracil at +2; much less strand separation is seen with uracil at +4. DNA unwinding has long been recognized as necessary for proof-reading exonuclease activity. The roles of M247 and Y261, amino acids suggested by structural studies to play a role in primer-template unwinding, have been probed. M247 appears to be unimportant, but 2-aminopurine fluorescence measurements show that Y261 plays a role in primer-template strand separation. Y261 is also required for full exonuclease activity and contributes to the fidelity of the polymerase. 相似文献
2.
Guillermo Pastor-Palacios Elisa Azuara-Liceaga Luis G. Brieba 《PLoS neglected tropical diseases》2010,4(8)
Background
Eukaryotic family A DNA polymerases are involved in mitochondrial DNA replication or translesion DNA synthesis. Here, we present evidence that the sole family A DNA polymerase from the parasite protozoan E. histolytica (EhDNApolA) localizes to the nucleus and that its biochemical properties indicate that this DNA polymerase may be involved in translesion DNA synthesis.Methodology and Results
EhDNApolA is the sole family A DNA polymerase in E. histolytica. An in silico analysis places family A DNA polymerases from the genus Entamoeba in a separate branch of a family A DNA polymerases phylogenetic tree. Biochemical studies of a purified recombinant EhDNApolA demonstrated that this polymerase is active in primer elongation, is poorly processive, displays moderate strand displacement, and does not contain 3′–5′ exonuclease or editing activity. Importantly, EhDNApolA bypasses thymine glycol lesions with high fidelity, and confocal microscopy demonstrates that this polymerase is translocated into the nucleus. These data suggest a putative role of EhDNApolA in translesion DNA synthesis in E. histolytica.Conclusion
This is the first report of the biochemical characterization of a DNA polymerase from E. histolytica. EhDNApolA is a family A DNA polymerase that is grouped into a new subfamily of DNA polymerases with translesion DNA synthesis capabilities similar to DNA polymerases from subfamily ν. 相似文献3.
Kei-ichi Takata Mercedes E. Arana Mineaki Seki Thomas A. Kunkel Richard D. Wood 《Nucleic acids research》2010,38(10):3233-3244
POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved ‘O-helix’ of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN. 相似文献
4.
Carolina B. Gabbai Joseph T. P. Yeeles Kenneth J. Marians 《The Journal of biological chemistry》2014,289(47):32811-32823
A number of different enzymatic pathways have evolved to ensure that DNA replication can proceed past template base damage. These pathways include lesion skipping by the replisome, replication fork regression followed by either correction of the damage and origin-independent replication restart or homologous recombination-mediated restart of replication downstream of the lesion, and bypass of the damage by a translesion synthesis DNA polymerase. We report here that of two translesion synthesis polymerases tested, only DNA polymerase IV, not DNA polymerase II, could engage productively with the Escherichia coli replisome to bypass leading strand template damage, despite the fact that both enzymes are shown to be interacting with the replicase. Inactivation of the 3′ → 5′ proofreading exonuclease of DNA polymerase II did not enable bypass. Bypass by DNA polymerase IV required its ability to interact with the β clamp and act as a translesion polymerase but did not require its “little finger” domain, a secondary region of interaction with the β clamp. Bypass by DNA polymerase IV came at the expense of the inherent leading strand lesion skipping activity of the replisome, indicating that they are competing reactions. 相似文献
5.
Tyler L. Dangerfield Serdal Kirmizialtin Kenneth A. Johnson 《The Journal of biological chemistry》2022,298(3)
Faithful replication of genomic DNA by high-fidelity DNA polymerases is crucial for the survival of most living organisms. While high-fidelity DNA polymerases favor canonical base pairs over mismatches by a factor of ∼1 × 105, fidelity is further enhanced several orders of magnitude by a 3′–5′ proofreading exonuclease that selectively removes mispaired bases in the primer strand. Despite the importance of proofreading to maintaining genome stability, it remains much less studied than the fidelity mechanisms employed at the polymerase active site. Here we characterize the substrate specificity for the proofreading exonuclease of a high-fidelity DNA polymerase by investigating the proofreading kinetics on various DNA substrates. The contribution of the exonuclease to net fidelity is a function of the kinetic partitioning between extension and excision. We show that while proofreading of a terminal mismatch is efficient, proofreading a mismatch buried by one or two correct bases is even more efficient. Because the polymerase stalls after incorporation of a mismatch and after incorporation of one or two correct bases on top of a mismatch, the net contribution of the exonuclease is a function of multiple opportunities to correct mistakes. We also characterize the exonuclease stereospecificity using phosphorothioate-modified DNA, provide a homology model for the DNA primer strand in the exonuclease active site, and propose a dynamic structural model for the transfer of DNA from the polymerase to the exonuclease active site based on MD simulations. 相似文献
6.
Henry J. Russell Tomas T. Richardson Kieran Emptage Bernard A. Connolly 《Nucleic acids research》2009,37(22):7603-7611
Archaeal family B polymerases bind tightly to the deaminated bases uracil and hypoxanthine in single-stranded DNA, stalling replication on encountering these pro-mutagenic deoxynucleosides four steps ahead of the primer–template junction. When uracil is specifically bound, the polymerase–DNA complex exists in the editing rather than the polymerization conformation, despite the duplex region of the primer-template being perfectly base-paired. In this article, the interplay between the 3′–5′ proofreading exonuclease activity and binding of uracil/hypoxanthine is addressed, using the family-B DNA polymerase from Pyrococcus furiosus. When uracil/hypoxanthine is bound four bases ahead of the primer–template junction (+4 position), both the polymerase and the exonuclease are inhibited, profoundly for the polymerase activity. However, if the polymerase approaches closer to the deaminated bases, locating it at +3, +2, +1 or even 0 (paired with the extreme 3′ base in the primer), the exonuclease activity is strongly stimulated. In these situations, the exonuclease activity is actually stronger than that seen with mismatched primer-templates, even though the deaminated base-containing primer-templates are correctly base-paired. The resulting exonucleolytic degradation of the primer serves to move the uracil/hypoxanthine away from the primer–template junction, restoring the stalling position to +4. Thus the 3′–5′ proofreading exonuclease contributes to the inability of the polymerase to replicate beyond deaminated bases. 相似文献
7.
The 3′→5′ exonuclease activity intrinsic to several DNA polymerases plays a primary role in genetic stability; it acts as a first line of defense in correcting DNA polymerase errors. A mismatched basepair at the primer terminus is the preferred substrate for the exonuclease activity over a correct basepair. The efficiency of the exonuclease as a proofreading activity for mispairs containing a DNA lesion varies, however, being dependent upon both the DNA polymerase/exonuclease and the type of DNA lesion. The exonuclease activities intrinsic to the T4 polymerase (family B) and DNA polymerase γ (family A) proofread DNA mispairs opposite endogenous DNA lesions, including alkylation, oxidation, and abasic adducts. However, the exonuclease of the Klenow polymerase cannot discriminate between correct and incorrect bases opposite alkylation and oxidative lesions. DNA damage alters the dynamics of the intramolecular partitioning of DNA substrates between the 3′→5′ exonuclease and polymerase activities. Enzymatic idling at lesions occurs when an exonuclease activity efficiently removes the same base that is preferentially incorporated by the DNA polymerase activity. Thus, the exonuclease activity can also act as a kinetic barrier to translesion synthesis (TLS) by preventing the stable incorporation of bases opposite DNA lesions. Understanding the downstream consequences of exonuclease activity at DNA lesions is necessary for elucidating the mechanisms of translesion synthesis and damage-induced cytotoxicity. 相似文献
8.
Promiscuous mismatch extension by human DNA polymerase lambda 总被引:2,自引:1,他引:1
Picher AJ García-Díaz M Bebenek K Pedersen LC Kunkel TA Blanco L 《Nucleic acids research》2006,34(11):3259-3266
DNA polymerase lambda (Pol λ) is one of several DNA polymerases suggested to participate in base excision repair (BER), in repair of broken DNA ends and in translesion synthesis. It has been proposed that the nature of the DNA intermediates partly determines which polymerase is used for a particular repair reaction. To test this hypothesis, here we examine the ability of human Pol λ to extend mismatched primer-termini, either on ‘open’ template-primer substrates, or on its preferred substrate, a 1 nt gapped-DNA molecule having a 5′-phosphate. Interestingly, Pol λ extended mismatches with an average efficiency of ≈10−2 relative to matched base pairs. The match and mismatch extension catalytic efficiencies obtained on gapped molecules were ≈260-fold higher than on template-primer molecules. A crystal structure of Pol λ in complex with a single-nucleotide gap containing a dG·dGMP mismatch at the primer-terminus (2.40 Å) suggests that, at least for certain mispairs, Pol λ is unable to differentiate between matched and mismatched termini during the DNA binding step, thus accounting for the relatively high efficiency of mismatch extension. This property of Pol λ suggests a potential role as a ‘mismatch extender’ during non-homologous end joining (NHEJ), and possibly during translesion synthesis. 相似文献
9.
Random mutagenesis constitutes an important approach for identifying critical regions of proteins, studying structure-function relations and developing novel proteins with desired properties. Perhaps, the most popular method is the error-prone PCR, in which mistakes are introduced into a gene, and hence a protein, during DNA polymerase-catalysed amplification cycles. Unfortunately, the relatively high fidelities of the thermostable DNA polymerases commonly used for PCR result in too few mistakes in the amplified DNA for efficient mutagenesis. In this paper, we describe mutants of the family B DNA polymerase from Pyrococcus furiosus (Pfu-Pol), with superb performance in error-prone PCR. The key amino acid changes occur in a short loop linking two long α-helices that comprise the ‘fingers’ sub-domain of the protein. This region is responsible for binding the incoming dNTPs and ensuring that only correct bases are inserted opposite the complementary base in the template strand. Mutations in the short loop, when combined with an additional mutation that abolishes the 3′–5′ proof-reading exonuclease activity, convert the extremely accurate wild-type polymerase into a variant with low fidelity. The mutant Pfu-Pols can be applied in error-prone PCR, under exactly the same conditions used for standard, high-fidelity PCR with the wild-type enzyme. Large quantities of amplified product, with a high frequency of nearly indiscriminate mutations, are produced. It is anticipated that the Pfu-Pol variants will be extremely useful for the randomization of gene, and hence protein, sequences. 相似文献
10.
The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I 总被引:10,自引:0,他引:10
K Bebenek C M Joyce M P Fitzgerald T A Kunkel 《The Journal of biological chemistry》1990,265(23):13878-13887
The fidelity of DNA synthesis by an exonuclease-proficient DNA polymerase results from the selectivity of the polymerization reaction and from exonucleolytic proofreading. We have examined the contribution of these two steps to the fidelity of DNA synthesis catalyzed by the large Klenow fragment of Escherichia coli DNA polymerase I, using enzymes engineered by site-directed mutagenesis to inactivate the proofreading exonuclease. Measurements with two mutant Klenow polymerases lacking exonuclease activity but retaining normal polymerase activity and protein structure demonstrate that the base substitution fidelity of polymerization averages one error for each 10,000 to 40,000 bases polymerized, and can vary more than 30-fold depending on the mispair and its position. Steady-state enzyme kinetic measurements of selectivity at the initial insertion step by the exonuclease-deficient polymerase demonstrate differences in both the Km and the Vmax for incorrect versus correct nucleotides. Exonucleolytic proofreading by the wild-type enzyme improves the average base substitution fidelity by 4- to 7-fold, reflecting efficient proofreading of some mispairs and less efficient proofreading of others. The wild-type polymerase is highly accurate for -1 base frameshift errors, with an error rate of less than or equal to 10(-6). The exonuclease-deficient polymerase is less accurate, suggesting that proofreading also enhances frameshift fidelity. Even without a proofreading exonuclease, Klenow polymerase has high frameshift fidelity relative to several other DNA polymerases, including eucaryotic DNA polymerase-alpha, an exonuclease-deficient, 4-subunit complex whose catalytic subunit is almost three times larger. The Klenow polymerase has a large (46 kDa) domain containing the polymerase active site and a smaller (22 kDa) domain containing the active site for the 3'----5' exonuclease. Upon removal of the small domain, the large polymerase domain has altered base substitution error specificity when compared to the two-domain but exonuclease-deficient enzyme. It is also less accurate for -1 base errors at reiterated template nucleotides and for a 276-nucleotide deletion error. Thus, removal of a protein domain of a DNA polymerase can affect its fidelity. 相似文献
11.
Effect of manganese on in vitro replication of damaged DNA catalyzed by the herpes simplex virus type-1 DNA polymerase 总被引:1,自引:1,他引:0
Villani G Tanguy Le Gac N Wasungu L Burnouf D Fuchs RP Boehmer PE 《Nucleic acids research》2002,30(15):3323-3332
In vitro bypass of damaged DNA by replicative DNA polymerases is usually blocked by helix-distorting or bulky DNA lesions. In this study, we report that substitution of the divalent metal ion Mg2+ with Mn2+ promotes quantitative replication of model DNA substrates containing the major cisplatin or N-2-acetylaminofluorene adducts by the catalytic subunit (UL30) of the replicative DNA polymerase of herpes simplex virus. The ability of Mn2+ ions to confer bypass of bulky lesions was not observed with other replicative DNA polymerases of the B family, such as bacteriophage T4 or δ polymerases. However, for these enzymes, manganese induced the incorporation of one nucleotide opposite the first (3′) guanine of the d(GpG) intrastrand cisplatin lesion. Translesion replication of the cisplatin adduct by UL30 led to the incorporation of mismatched bases, with the preferential incorporation of dAMP opposite the 3′ guanine of the lesion. Furthermore, substitution of MgCl2 with MnCl2 greatly inhibited the 3′ to 5′ exonuclease of UL30 but had a far lesser effect on that of T4 DNA polymerase. Finally, manganese induced a conformational change in the structure of UL30 bound to the platinated substrate. Taken together, the latter findings suggest a mechanism by which manganese might allow UL30 to efficiently promote translesion DNA synthesis in vitro. 相似文献
12.
Jung-Hoon Yoon Jayati Roy Choudhury Jeseong Park Satya Prakash Louise Prakash 《The Journal of biological chemistry》2014,289(19):13177-13185
The biological functions of human DNA polymerase (pol) θ, an A family polymerase, have remained poorly defined. Here we identify a role of polθ in translesion synthesis (TLS) in human cells. We show that TLS through the thymine glycol (TG) lesion, the most common oxidation product of thymine, occurs via two alternative pathways, in one of which, polymerases κ and ζ function together and mediate error-free TLS, whereas in the other, polθ functions in an error-prone manner. Human polθ is comprised of an N-terminal ATPase/helicase domain, a large central domain, and a C-terminal polymerase domain; however, we find that only the C-terminal polymerase domain is required for TLS opposite TG in human cells. In contrast to TLS mediated by polκ and polζ, in which polζ would elongate the chain from the TG:A base pair formed by polκ action, the ability of polθ alone to carry out the nucleotide insertion step, as well as the subsequent extension step that presents a considerable impediment due to displacement of the 5′ template base, suggests that the polθ active site can accommodate highly distorting DNA lesions. 相似文献
13.
[目的]以嗜酸嗜热硫化叶菌Sulfolobus acidocaldarius的DNA聚合酶IV (Saci_0554)为例,表征其跨越模板上损伤碱基的DNA合成效果。[方法]将DNA聚合酶IV (SacpolIV)在大肠杆菌中进行重组表达,经亲和层析纯化得到SacpolIV蛋白;利用人工合成的带有不同损伤的寡核苷酸片段作为模板DNA,用尿素变性聚丙烯酰胺凝胶电泳技术,鉴定SacpolIV在体外跨越各种损伤碱基进行跨损伤合成的催化能力。[结果]SacpolIV重组蛋白能够不同程度地跨越嘌呤和嘧啶损伤,跨越能力的高低取决于损伤碱基与正常碱基形成氢键的能力。本研究还发现,SacpolIV能够在DNA链中掺入核糖核苷酸,但掺入核糖核苷酸的效率低于脱氧核糖核苷酸。[结论]本研究证实SacpolIV具有很强的跨越损伤合成能力,能够跨越多种氢键配对能力减弱的损伤碱基,为其在细胞内的跨越损伤合成功能提供了生化证据。 相似文献
14.
The balance between exonuclease and polymerase activities promotes DNA synthesis over degradation when nucleotides are correctly added to the new strand by replicative B-family polymerases. Misincorporations shift the balance toward the exonuclease site, and the balance tips back in favor of DNA synthesis when the incorrect nucleotides have been removed. Most B-family DNA polymerases have an extended β-hairpin loop that appears to be important for switching from the exonuclease site to the polymerase site, a process that affects fidelity of the DNA polymerase. Here, we show that DNA polymerase ε can switch between the polymerase site and exonuclease site in a processive manner despite the absence of an extended β-hairpin loop. K967 and R988 are two conserved amino acids in the palm and thumb domain that interact with bases on the primer strand in the minor groove at positions n−2 and n−4/n−5, respectively. DNA polymerase ε depends on both K967 and R988 to stabilize the 3′-terminus of the DNA within the polymerase site and on R988 to processively switch between the exonuclease and polymerase sites. Based on a structural alignment with DNA polymerase δ, we propose that arginines corresponding to R988 might have a similar function in other B-family polymerases. 相似文献
15.
Human DNA polymerase iota utilizes different nucleotide incorporation mechanisms dependent upon the template base
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Human DNA polymerase ι (Polι) is a member of the Y family of DNA polymerases involved in translesion DNA synthesis. Polι is highly unusual in that it possesses a high fidelity on template A, but has an unprecedented low fidelity on template T, preferring to misincorporate a G instead of an A. To understand the mechanisms of nucleotide incorporation opposite different template bases by Polι, we have carried out pre-steady-state kinetic analyses of nucleotide incorporation opposite templates A and T. These analyses have revealed that opposite template A, the correct nucleotide is preferred because it is bound tighter and is incorporated faster than the incorrect nucleotides. Opposite template T, however, the correct and incorrect nucleotides are incorporated at very similar rates, and interestingly, the greater efficiency of G misincorporation relative to A incorporation opposite T arises predominantly from the tighter binding of G. Based on these results, we propose that the incipient base pair is accommodated differently in the active site of Polι dependent upon the template base and that when T is the templating base, Polι accommodates the wobble base pair better than the Watson-Crick base pair. 相似文献
16.
Vertebrate DNA damage tolerance requires the C-terminus but not BRCT or transferase domains of REV1 总被引:6,自引:1,他引:5
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REV1 is central to the DNA damage response of eukaryotes through an as yet poorly understood role in translesion synthesis. REV1 is a member of the Y-type DNA polymerase family and is capable of in vitro deoxycytidyl transferase activity opposite a range of damaged bases. However, non-catalytic roles for REV1 have been suggested by the Saccharomyces cerevisiae rev1-1 mutant, which carries a point mutation in the N-terminal BRCT domain, and the recently demonstrated ability of the mammalian protein to interact with each of the other translesion polymerases via its extreme C-terminus. Here, we show that a region adjacent to this polymerase interacting domain mediates an interaction with PCNA. These C-terminal domains of REV1 are necessary, although not sufficient, for effective tolerance of DNA damage in the avian cell line DT40, while the BRCT domain and transferase activity are not directly required. Together these data provide strong support for REV1 playing an important non-catalytic role in coordinating translesion synthesis. Further, unlike in budding yeast, rad18 is not epistatic to rev1 for DNA damage tolerance suggesting that REV1 and RAD18 play largely independent roles in the control of vertebrate translesion synthesis. 相似文献
17.
Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase κ (Polκ) encoded by the DINB1 gene. Our recent studies have indicated that human Polκ is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Polκ also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Polκ during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Polκ synthesizes DNA with extraordinarily low fidelity. At the lacZα target gene, human Polκ made on average one error for every 200 nucleotides synthesized, with a predominant T→G transversion mutation at a rate of 1/147. The overall error rate of human Polκ is 1.7-fold lower than human Polη, but 33-fold higher than human Polβ, a DNA polymerase with very low fidelity. Thus, human Polκ is one of the most inaccurate DNA polymerases known. These results support a role for human Polκ in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage. 相似文献
18.
Solid-phase synthesis of oligopurine deoxynucleic guanidine (DNG) and analysis of binding with DNA oligomers
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The first stepwise solid-phase synthesis of deoxynucleic guanidine (DNG), a positively charged DNA analog, using controlled pore glass as the solid support is reported. For the first time, purine bases have been incorporated into the DNG oligomer and DNG has been synthesized using a solid-phase method, proceeding in the 3′→5′ direction, that is compatible with the cleavage conditions used in the solid-phase synthesis of DNA. A DNG sequence containing a pentameric tract of adenosine nucleosides has been synthesized and the thermal denaturation temperature of its complexes with complementary thymidyl DNA oligomers was 79°C. Binding of thymidyl DNA oligomers to adenyl DNG oligomers is 2:1, as seen in thymidyl and adenyl DNA triplexes. No binding of adenyl DNG with octameric cytidyl DNA was observed, indicating that the positive charge does not overcome base pairing fidelity. 相似文献
19.
Katherine A. Donigan Mary P. McLenigan Wei Yang Myron F. Goodman Roger Woodgate 《The Journal of biological chemistry》2014,289(13):9136-9145
Accurate DNA synthesis in vivo depends on the ability of DNA polymerases to select dNTPs from a nucleotide pool dominated by NTPs. High fidelity replicative polymerases have evolved to efficiently exclude NTPs while copying long stretches of undamaged DNA. However, to bypass DNA damage, cells utilize specialized low fidelity polymerases to perform translesion DNA synthesis (TLS). Of interest is human DNA polymerase ι (pol ι), which has been implicated in TLS of oxidative and UV-induced lesions. Here, we evaluate the ability of pol ι to incorporate NTPs during DNA synthesis. pol ι incorporates and extends NTPs opposite damaged and undamaged template bases in a template-specific manner. The Y39A “steric gate” pol ι mutant is considerably more active in the presence of Mn2+ compared with Mg2+ and exhibits a marked increase in NTP incorporation and extension, and surprisingly, it also exhibits increased dNTP base selectivity. Our results indicate that a single residue in pol ι is able to discriminate between NTPs and dNTPs during DNA synthesis. Because wild-type pol ι incorporates NTPs in a template-specific manner, certain DNA sequences may be “at risk” for elevated mutagenesis during pol ι-dependent TLS. Molecular modeling indicates that the constricted active site of wild-type pol ι becomes more spacious in the Y39A variant. Therefore, the Y39A substitution not only permits incorporation of ribonucleotides but also causes the enzyme to favor faithful Watson-Crick base pairing over mutagenic configurations. 相似文献
20.
Marc J. Prindle Michael W. Schmitt Fabio Parmeggiani Lawrence A. Loeb 《The Journal of biological chemistry》2013,288(8):5572-5580
DNA polymerase δ (Pol δ) is one of the major replicative DNA polymerases in eukaryotic cells, catalyzing lagging strand synthesis as well as playing a role in many DNA repair pathways. The catalytic site for polymerization consists of a palm domain and mobile fingers domain that opens and closes each catalytic cycle. We explored the effect of amino acid substitutions in a region of the highly conserved sequence motif B in the fingers domain on replication fidelity. A novel substitution, A699Q, results in a marked increase in mutation rate at the yeast CAN1 locus, and is synthetic lethal with both proofreading deficiency and mismatch repair deficiency. Modeling the A699Q mutation onto the crystal structure of Saccharomyces cerevisiae Pol δ template reveals four potential contacts for A699Q but not for A699. We substituted alanine for each of these residues and determined that an interaction with multiple residues of the N-terminal domain is responsible for the mutator phenotype. The corresponding mutation in purified human Pol δ results in a similar 30-fold increase in mutation frequency when copying gapped DNA templates. Sequence analysis indicates that the most characteristic mutation is a guanine-to-adenine (G to A) transition. The increase in deoxythymidine 5′-triphosphate-G mispairs was confirmed by performing steady state single nucleotide addition studies. Our combined data support a model in which the Ala-to-Gln substitution in the fingers domain of Pol δ results in an interaction with the N-terminal domain that affects the base selectivity of the enzyme. 相似文献