Showing papers on "DNA clamp published in 2000"
TL;DR: A novel method that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions that employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.
Abstract: We have developed a novel method, termed loop-mediated isothermal amplification (LAMP), that amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions. This method employs a DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA. An inner primer containing sequences of the sense and antisense strands of the target DNA initiates LAMP. The following strand displacement DNA synthesis primed by an outer primer releases a single-stranded DNA. This serves as template for DNA synthesis primed by the second inner and outer primers that hybridize to the other end of the target, which produces a stem–loop DNA structure. In subsequent LAMP cycling one inner primer hybridizes to the loop on the product and initiates displacement DNA synthesis, yielding the original stem–loop DNA and a new stem–loop DNA with a stem twice as long. The cycling reaction continues with accumulation of 109 copies of target in less than an hour. The final products are stem–loop DNAs with several inverted repeats of the target and cauliflower-like structures with multiple loops formed by annealing between alternately inverted repeats of the target in the same strand. Because LAMP recognizes the target by six distinct sequences initially and by four distinct sequences afterwards, it is expected to amplify the target sequence with high selectivity.
6,765 citations
TL;DR: It is shown here that the central DNA flap acts as a cis-determinant of HIV-1 DNA nuclear import, which can be corrected to wild-type levels by reinsertion of the DNA flap sequence.
929 citations
TL;DR: The initial steps common to these recombination and recombination-dependent replication processes are reviewed and the machinery of homologous recombination acts at these breaks and gaps to promote the events that result in gene recombination.
704 citations
TL;DR: Structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APe1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.
Abstract: Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.
651 citations
TL;DR: It is reported that a plasmid replicating in Xenopus egg extracts becomes negatively supercoiled during replication initiation, indicating that the helicase that unwinds DNA during initiation can become uncoupled from the replication fork.
445 citations
TL;DR: Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle, and finds a force-induced 100-fold increase in exonucleolysis above 40 pN.
Abstract: T7 DNA polymerase1,2 catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3′→5′ exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a ‘right hand’ shape which is common to most polymerases with fingers, palm and thumb domains3,4. The rate-limiting step for replication is thought to involve a conformational change between an ‘open fingers’ state in which the active site samples nucleotides, and a ‘closed’ state in which nucleotide incorporation occurs3,5. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of ∼34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.
441 citations
01 Jan 2000
TL;DR: In this paper, a single-molecule assay based on the differential elasticity of single-stranded and doublestranded DNA was used to show that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of ∼34
Abstract: T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3′→5′ exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a ‘right hand’ shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an ‘open fingers’ state in which the active site samples nucleotides, and a ‘closed’ state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of ∼34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.
401 citations
TL;DR: The low fidelity of hPolη may derive from a flexible active site that renders the enzyme more tolerant of geometric distortions in DNA and enables it to synthesize DNA past a T-T dimer.
388 citations
TL;DR: It is shown that human pol-η copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied, indicating a relaxed requirement for correct base pairing geometry and indicates that the function of pol- η may be tightly controlled to prevent potentially mutagenic DNA synthesis.
Abstract: A superfamily of DNA polymerases that bypass lesions in DNA has been described. Some family members are described as error-prone because mutations that inactivate the polymerase reduce damage-induced mutagenesis. In contrast, mutations in the skin cancer susceptibility gene XPV, which encodes DNA polymerase (pol)-eta, lead to increased ultraviolet-induced mutagenesis. This, and the fact that pol-eta primarily inserts adenines during efficient bypass of thymine-thymine dimers in vitro, has led to the description of pol-eta as error-free. However, here we show that human pol-eta copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied. Pol-eta lacks an intrinsic proofreading exonuclease activity and, depending on the mismatch, makes one base substitution error for every 18 to 380 nucleotides synthesized. This very low fidelity indicates a relaxed requirement for correct base pairing geometry and indicates that the function of pol-eta may be tightly controlled to prevent potentially mutagenic DNA synthesis.
375 citations
TL;DR: A ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level suggests the existence of efficient repair pathways for removal of topoisomersase I-DNA covalent adducts in ribosomal DNA.
Abstract: Topoisomerase I cleavage complexes can be induced by a variety of DNA damages and by the anticancer drug camptothecin. We have developed a ligation-mediated PCR (LM-PCR) assay to analyze replication-mediated DNA double-strand breaks induced by topoisomerase I cleavage complexes in human colon carcinoma HT29 cells at the nucleotide level. We found that conversion of topoisomerase I cleavage complexes into replication-mediated DNA double-strand breaks was only detectable on the leading strand for DNA synthesis, which suggests an asymmetry in the way that topoisomerase I cleavage complexes are metabolized on the two arms of a replication fork. Extension by Taq DNA polymerase was not required for ligation to the LM-PCR primer, indicating that the 3' DNA ends are extended by DNA polymerase in vivo closely to the 5' ends of the topoisomerase I cleavage complexes. These findings suggest that the replication-mediated DNA double-strand breaks generated at topoisomerase I cleavage sites are produced by replication runoff. We also found that the 5' ends of these DNA double-strand breaks are phosphorylated in vivo, which suggests that a DNA 5' kinase activity acts on the double-strand ends generated by replication runoff. The replication-mediated DNA double-strand breaks were rapidly reversible after cessation of the topoisomerase I cleavage complexes, suggesting the existence of efficient repair pathways for removal of topoisomerase I-DNA covalent adducts in ribosomal DNA.
342 citations
TL;DR: Yeast and human POLη replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair, suggesting an additional role for Polη in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-xoG in DNA.
Abstract: Oxidative damage to DNA has been proposed to have a role in cancer and ageing1. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed2,3. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion4; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions5. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. In Saccharomyces cerevisiae, deletion of OGG1, encoding a DNA glycosylase that functions in the removal of 8-oxoG when paired with C, causes an increase in the rate of G:C to T:A transversions6. The ogg1Δ msh2Δ double mutant displays a higher rate of CAN1S to can1r forward mutations than the ogg1Δ or msh2Δ single mutants, and this enhanced mutagenesis is primarily due to G:C to T:A transversions7. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Polη, that efficiently replicates DNA containing a cis-syn thymine-thymine (T-T) dimer by inserting two adenines across from the dimer8. In humans, mutations in the yeast RAD30 counterpart, POLH, cause the variant form of xeroderma pigmentosum9,10 (XP-V), and XP-V individuals suffer from a high incidence of sunlight-induced skin cancers. Here we show that yeast and human POLη replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this base pair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POLη in the yeast ogg1Δ mutant. Our results suggest an additional role for Polη in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.
TL;DR: The replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limiting step of each cycle, consistent with the induced-fit mechanism for error detection during replication.
Abstract: A new approach to the study of DNA/protein interactions has been opened through the recent advances in the manipulation of single DNA molecules. These allow the behavior of individual molecular motors to be studied under load and compared with bulk measurements. One example of such a motor is the DNA polymerase, which replicates DNA. We measured the replication rate by a single enzyme of a stretched single strand of DNA. The marked difference between the elasticity of single- and double-stranded DNA allows for the monitoring of replication in real time. We have found that the rate of replication depends strongly on the stretching force applied to the template. In particular, by varying the load we determined that the biochemical steps limiting replication are coupled to movement. The replication rate increases at low forces, decreases at forces greater than 4 pN, and ceases when the single-stranded DNA substrate is under a load greater than ≈20 pN. The decay of the replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limiting step of each cycle. This observation is consistent with the induced-fit mechanism for error detection during replication.
TL;DR: A molecular model for Rad17(Sp), similar to the subunits of the RFC clamp-loading complex, is concluded that members of this family are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA.
Abstract: The repair of damaged DNA is coupled to the completion of DNA replication by several cell cycle checkpoint proteins, including, for example, in fission yeast Rad1Sp, Hus1Sp, Rad9Sp and Rad17Sp. We have found that these four proteins are conserved with protein sequences throughout eukaryotic evolution. Using computational techniques, including fold recognition, comparative modeling and generalized sequence profiles, we have made high confidence structure predictions for the each of the Rad1, Hus1 and Rad9 protein families (Rad17Sc, Mec3Sc and Ddc1Sc in budding yeast, respectively). Each of these families was found to share a common protein fold with that of PCNA, the sliding clamp protein that tethers DNA polymerase to its template. We used previously reported genetic and biochemical data for these proteins from yeast and human cells to predict a heterotrimeric PCNA-like ring structure for the functional Rad1/Rad9/Hus1 complex and to determine their exact order within it. In addition, for each individual protein family, contact regions with neighbors within the PCNA-like ring were identified. Based on a molecular model for Rad17Sp, we concluded that members of this family, similar to the subunits of the RFC clamp-loading complex, are capable of coupling ATP binding with conformational changes required to load a sliding clamp onto DNA. This model substantiates previous findings regarding the behavior of Rad17 family proteins upon DNA damage and within the RFC complex of clamp-loading proteins.
TL;DR: It is reported that the DINB1 gene encodes a DNA polymerase (designated polkappa), which incorporates mismatched bases on a nondamaged template with a high frequency, which implicate an important role in the mutagenic bypass of certain types of DNA lesions.
Abstract: The Escherichia coli protein DinB is a newly identified error-prone DNA polymerase. Recently, a human homolog of DinB was identified and named DINB1. We report that the DINB1 gene encodes a DNA polymerase (designated polkappa), which incorporates mismatched bases on a nondamaged template with a high frequency. Moreover, polkappa bypasses an abasic site and N-2-acetylaminofluorene (AAF)-adduct in an error-prone manner but does not bypass a cis-syn or (6-4) thymine-thymine dimer or a cisplatin-adduct. Therefore, our results implicate an important role for polkappa in the mutagenic bypass of certain types of DNA lesions.
TL;DR: This corrects the article DOI: 10.1038/35000249 to indicate that the author of the paper is a doctor of medicine rather than a scientist, as previously reported.
Abstract: Nature 403, 451–456 (2000) The title of this Letter contained an error. The correct title is as printed above.
TL;DR: It is suggested that DNA-PKcs-containing complexes formed at DNA ends enhance the association of DNA ends via protein-protein interactions, thereby stimulating intermolecular ligation.
TL;DR: It is proposed that B. subtilis, and probably many bacteria, harness energy released during nucleotide condensation by a stationary replisome to facilitate chromosome partitioning.
TL;DR: The very low fidelity and moderate processivity of pol κ is novel in comparison to any previously studied DNA polymerase, and is consistent with a role in spontaneous mutagenesis.
TL;DR: The number of known DNA polymerases has increased to at least nine (called alpha, beta, gamma, delta, epsilon, zeta, eta, t and iota), and yeast Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase.
TL;DR: A specific interaction of Rad54 with the Rad51-ssDNA complex enhances the ability of the former to remodel DNA and allows the latter to harvest the negative supercoils generated for DNA joint formation.
TL;DR: In this article, the authors synthesize DNA substrates containing the expected gap and 5′ two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro.
Abstract: Diverse mobile DNA elements are believed to pirate host cell enzymes to complete DNA transfer. Prominent examples are provided by retroviral cDNA integration and transposon insertion. These reactions initially involve the attachment of each element 3′ DNA end to staggered sites in the host DNA by element-encoded integrase or transposase enzymes. Unfolding of such intermediates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesized DNA substrates containing the expected gap and 5′ two-base flap structure present in retroviral integration intermediates and tested candidate enzymes for the ability to support repair in vitro. We find three required activities, two of which can be satisfied by multiple enzymes. These are a polymerase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, III, or IV and its cofactor XRCC4). A proposed pathway involving retroviral integrase and reverse transcriptase did not carry out repair under the conditions tested. In addition, prebinding of integrase protein to gapped DNA inhibited repair reactions, indicating that gap repair in vivo may require active disassembly of the integrase complex.
TL;DR: The data indicate that polι possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.
Abstract: Human DNA polymerase ι (polι) is a recently discovered enzyme that exhibits extremely low fidelity on undamaged DNA templates. Here, we show that polι is able to facilitate limited translesion replication of a thymine–thymine cyclobutane pyrimidine dimer (CPD). More importantly, however, the bypass event is highly erroneous. Gel kinetic assays reveal that polι misinserts T or G opposite the 3′ T of the CPD ∼1.5 times more frequently than the correct base, A. While polι is unable to extend the T·T mispair significantly, the G·T mispair is extended and the lesion completely bypassed, with the same efficiency as that of the correctly paired A·T base pair. By comparison, polι readily misinserts two bases opposite a 6-4 thymine–thymine pyrimidine–pyrimidone photoproduct (6-4PP), but complete lesion bypass is only a fraction of that observed with the CPD. Our data indicate, therefore, that polι possesses the ability to insert nucleotides opposite UV photoproducts as well as to perform unassisted translesion replication that is likely to be highly mutagenic.
TL;DR: It is shown here that 5‐methylcytosine occurs in the DNA of D.melanogaster at a level of ∼1 in 1000–2000 cytosine residues in adult flies.
Abstract: It is commonly accepted that the DNA of Drosophila melanogaster does not contain 5-methylcytosine, which is essential in the development of most eukaryotes. We have developed a new, highly specific and sensitive assay to detect the presence of 5-methylcytosine in genomic DNA. The DNA is degraded to nucleosides, 5-methylcytosine purified by HPLC and, for detection by 1D- and 2D-TLC, radiolabeled using deoxynucleoside kinase and [γ-32P]ATP. Using this assay, we show here that 5-methylcytosine occurs in the DNA of D.melanogaster at a level of ∼1 in 1000–2000 cytosine residues in adult flies. DNA methylation is detectable in all stages of D.melanogaster development.
TL;DR: These findings demonstrate that melanin is a potent inhibitor of thermostable DNA polymerase in vitro and that the inhibitory effect is conferred by a direct and reversible polymerase-melanin interaction.
TL;DR: The ninth human DNA polymerase, Polι, encoded by the RAD30B gene is described and it is shown that human Polι violates the Watson-Crick base-pairing rule opposite template T, suggesting that DNA Polι may additionally play a specialized function in human biology.
Abstract: DNA polymerase activity is essential for replication, recombination, repair, and mutagenesis. All DNA polymerases studied so far from any biological source synthesize DNA by the Watson-Crick base-pairing rule, incorporating A, G, C, and T opposite the templates T, C, G, and A, respectively. Non-Watson-Crick base pairs would lead to mutations. In this report, we describe the ninth human DNA polymerase, Polι, encoded by the RAD30B gene. We show that human Polι violates the Watson-Crick base-pairing rule opposite template T. During base selection, human Polι preferred T-G base pairing, leading to G incorporation opposite template T. The resulting T-G base pair was less efficiently extended by human Polι compared to the Watson-Crick base pairs. Consequently, DNA synthesis frequently aborted opposite template T, a property we designated the T stop. This T stop restricted human Polι to a very short stretch of DNA synthesis. Furthermore, kinetic analyses show that human Polι copies template C with extraordinarily low fidelity, misincorporating T, A, and C with unprecedented frequencies of 1/9, 1/10, and 1/11, respectively. Human Polι incorporated one nucleotide opposite a template abasic site more efficiently than opposite a template T, suggesting a role for human Polι in DNA lesion bypass. The unique features of preferential G incorporation opposite template T and T stop suggest that DNA Polι may additionally play a specialized function in human biology.
TL;DR: It is shown that Mre11 exonuclease activity is sensitive to the presence of heterologous DNA, and to the structure and sequence of its ends, which means that addition of mismatched DNA ends stimulates degradation of DNA by Mre 11, whereas cohesive ends strongly inhibit it.
Abstract: Repair of DNA double-strand breaks in vertebrate cells occurs mainly by an end-joining process that often generates junctions with sequence homologies of a few nucleotides. Mre11 is critical for this mode of repair in budding yeast and has been implicated in the microhomology-based joining. Here, we show that Mre11 exonuclease activity is sensitive to the presence of heterologous DNA, and to the structure and sequence of its ends. Addition of mismatched DNA ends stimulates degradation of DNA by Mre11, whereas cohesive ends strongly inhibit it. Furthermore, if a sequence identity is revealed during the course of degradation, it causes Mre11 nuclease activity to pause, thus stabilizing the junction at a site of microhomology. A nuclease-deficient Mre11 mutant that still binds DNA can also stimulate degradation by wild-type Mre11, suggesting that Mre11-DNA complexes may interact to bridge DNA ends and facilitate DNA joining.
TL;DR: Results show that human Pol(eta) is capable of error-prone translesion DNA syntheses in vitro and suggest that Pol (eta) may bypass certain lesions with a mutagenic consequence in humans.
Abstract: DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase η (Polη), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Polη, we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Polη efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Polη effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant –1 deletion was also observed when the template base 5′ to the abasic site is a T. Human Polη partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N2-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N2-dG lesion in mammalian cells. These results show that human Polη is capable of error-prone translesion DNA syntheses in vitro and suggest that Polη may bypass certain lesions with a mutagenic consequence in humans.
TL;DR: It is shown here that WRN functionally interacts with DNA polymerase delta (pol delta), a eukaryotic polymerase required for DNA replication and DNA repair, and that disruption of WRN-pol delta interaction in WS cells may contribute to the previously observed S-phase defects and/or the unusual sensitivity to a limited number of DNA damaging agents.
Abstract: Werner Syndrome (WS) is an inherited disease characterized by premature onset of aging, increased cancer incidence, and genomic instability. The WS gene encodes a 1,432-amino acid polypeptide (WRN) with a central domain homologous to the RecQ family of DNA helicases. Purified WRN unwinds DNA with 3′→5′ polarity, and also possesses 3′→5′ exonuclease activity. Elucidation of the physiologic function(s) of WRN may be aided by the identification of WRN-interacting proteins. We show here that WRN functionally interacts with DNA polymerase δ (pol δ), a eukaryotic polymerase required for DNA replication and DNA repair. WRN increases the rate of nucleotide incorporation by pol δ in the absence of proliferating cell nuclear antigen (PCNA) but does not stimulate the activity of eukaryotic DNA polymerases α or ɛ, or a variety of other DNA polymerases. Moreover, we show that functional interaction with WRN is mediated through the third subunit of pol δ: i.e., Pol32p of Saccharomyces cerevisae, corresponding to the recently identified p66 subunit of human pol δ. Absence of the third subunit abrogates stimulation by WRN, and stimulation is restored by reconstituting the three-subunit enzyme. Our findings suggest that WRN may facilitate pol δ-mediated DNA replication and/or DNA repair and that disruption of WRN-pol δ interaction in WS cells may contribute to the previously observed S-phase defects and/or the unusual sensitivity to a limited number of DNA damaging agents.
01 Jan 2000
TL;DR: The structure of the Escherichia coli AlkA complex offers the first glimpse of a helix-hairpin-helix (HhH) glycosylase complexed to DNA as mentioned in this paper.
Abstract: The Escherichia coli AlkA protein is a base excision repair glycosylase that removes a variety of alkylated bases from DNA. The 2.5 A crystal structure of AlkA complexed to DNA shows a large distortion in the bound DNA. The enzyme flips a 1-azaribose abasic nucleotide out of DNA and induces a 66° bend in the DNA with a marked widening of the minor groove. The position of the 1-azaribose in the enzyme active site suggests an S N1-type mechanism for the glycosylase reaction, in which the essential catalytic Asp238 provides direct assistance for base removal. Catalytic selectivity might result from the enhanced stacking of positively charged, alkylated bases against the aromatic side chain of Trp272 in conjunction with the relative ease of cleaving the weakened glycosylic bond of these modified nucleotides. The structure of the AlkA–DNA complex offers the first glimpse of a helix–hairpin– helix (HhH) glycosylase complexed to DNA. Modeling studies suggest that other HhH glycosylases can bind to DNA in a similar manner.
TL;DR: The human DINB1 gene shares a high degree of homology with the Escherichia coli dinB gene, and the hDinB1-encoded protein is purify and shown to be a DNA polymerase, which is named hPoltheta because it is unable to bypass a cis-syn thymine-thymine dimer.
Abstract: The human DINB1 gene shares a high degree of homology with the Escherichia coli dinB gene. Here, we purify the hDINB1-encoded protein and show that it is a DNA polymerase. Because hDinB1 is the eighth eukaryotic DNA polymerase to be described, we have named it DNA polymerase (Pol) θ. hPolθ is unable to bypass a cis-syn thymine–thymine dimer, nor does it bypass a (6–4) photoproduct or an abasic site. We also examine the fidelity of hPolθ on nondamaged DNA templates by steady-state kinetic analyses and find that hPolθ misincorporates deoxynucleotides with a frequency of about 10−3 to 10−4. We discuss the relationship between the fidelity of hPolθ and its inability to bypass DNA damage.