Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation.     

极端嗜热古菌DNA修复核酸内切酶的研究进展

极端嗜热古菌由于生活在高温环境,其基因组DNA面临着严重的挑战,因此,它们如何维持其基因组稳定是本研究领域最为关注的科学问题之一。极端嗜热古菌具有与常温微生物相似的自发突变频率,暗示着它们比常温微生物具有更加有效的DNA修复体系进行修复高温所造成的基因组DNA损伤。目前,极端嗜热古菌DNA修复的分子机制尚不清楚。核酸内切酶在DNA修复途径中发挥着重要的作用。基因组序列显示极端嗜热古菌编码多种DNA修复核酸内切酶,但是其研究尚处于初期阶段。本文综述了极端嗜热古菌DNA修复核酸内切酶Nuc S、Endo V、Endo Q、XPF和Hjc的研究进展,并对今后的研究提出了展望。     

Inhibition of translesion DNA polymerase by archaeal reverse gyrase

Reverse gyrase is a unique DNA topoisomerase endowed with ATP-dependent positive supercoiling activity. It is typical of microorganisms living at high temperature and might play a role in maintenance of genome stability and repair. We have identified the translesion DNA polymerase SsoPolY/Dpo4 as one partner of reverse gyrase in the hyperthermophilic archaeon Sulfolobus solfataricus. We show here that in cell extracts, PolY and reverse gyrase co-immunoprecipitate with each other and with the single strand binding protein, SSB. The interaction is confirmed in vitro by far-western and Surface Plasmon Resonance. In functional assays, reverse gyrase inhibits PolY, but not the S. solfataricus B-family DNA polymerase PolB1. Mutational analysis shows that inhibition of PolY activity depends on both ATPase and topoisomerase activities of reverse gyrase, suggesting that the intact positive supercoiling activity is required for PolY inhibition. In vivo, reverse gyrase and PolY are degraded after induction of DNA damage. Inhibition by reverse gyrase and degradation might act as a double mechanism to control PolY and prevent its potentially mutagenic activity when undesired. Inhibition of a translesion polymerase by topoisomerase-induced modification of DNA structure may represent a previously unconsidered mechanism of regulation of these two-faced enzymes.     

Modulation by polyamines of DNA-dependent DNA polymerase activity from human serum

1. Spermine, spermidine and putrescine activated DNA-dependent DNA polymerase from human sera by 47-125% at the concentrations of 0.2, 3 and 30 mM, respectively. 2. The polyamines shifted the optimal MgCl2 concentration for the polymerase activity from 10 mM to more physiological 5 mM. 3. Histamine having amino and imino groups at both ends of the molecule also increased the DNA polymerase activity, while cyclopentylamine and n-butylamine showed no effects on the enzyme activity. 4. The stimulatory effect of polyamines on the DNA polymerase activity was more evident with poly(dC)p(dG) used as a template/primer than with poly(dA)p(dT).     

Multiple DNA polymerase activity solubilized from higher plant chromatin

DNA polymerase preparations solubilized from chromatin of unwashed and washed sugar beet storage tissue exhibit multiple activity peaks when separated by DEAE-Sephadex chromatography. Activity peaks isolated from unwashed and washed tissue required Mg²⁺, all four deoxynucleoside triphosphates and added DNA. Activity from peaks isolated from washed tissue was completely sensitive to pancreatic DNase and insensitive to RNase. Enzyme activity was increased in the tissue during the washing period. In addition, there was a drastic change in template specificity from single-stranded primer in unwashed tissue to double-stranded DNA in washed tissue.     

A cell-cycle-dependent DNA polymerase activity that replicates intact DNA in chromatin

An insoluble DNA polymerase activity that replicates the intact chromatin template at 85% of the rate found in vivo has been partially characterized. HeLa cells, encapsulated in agarose microbeads, are lysed using an isotonic salt concentration: the resulting encapsulated nuclei contain polymerase associated with a nucleoskeleton and the unbroken template. This preparation can be manipulated freely without aggregation or breaking the DNA and yet is accessible to enzymes and other probes. The major activity, which is sensitive to aphidicolin, is found only in S-phase nuclei and replicates DNA semi-conservatively, forming intermediates that are ligated efficiently into larger products.     

Stimulation of DNA polymerase alpha activity by microtubule-associated proteins.

Microtubule-associated protein 2 (MAP2) isolated from porcine brains stimulated the activity of DNA polymerase alpha immunopurified from calf thymus or human lymphoma cells, in a dose-dependent manner. This stimulation was pronounced when activated DNA or poly(dA).(dT)10 was used as the template-primer. DNA polymerase alpha bound to a MAP2-immobilized column, whereas preincubation of the enzyme with unbound MAP2 prevented binding to the column. These events suggested that a physical binding occurred between the polymerase and MAP2. Kinetic analyses revealed that MAP2 decreased the Km value of the polymerase for deoxyribonucleotides, irrespective of the species of template-primer. A concomitant increase in Vmax was observed; however, the extent of the increase depended on the species of template-primer. MAP2 also decreased the Km value of the polymerase for template-primers when activated DNA of poly(dA).(dT)10 was used as the template-primer. Product analyses showed that MAP2 did not significantly alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis. The stimulation by MAP2 occurred specifically in the activity of DNA polymerase alpha, but not DNA polymerases beta, gamma, and I from Escherichia coli. Other MAPs, tau and 190-kDa MAP, could substitute for MAP2. Thus, the specific stimulation of DNA polymerase alpha by MAPs supports the notion of a possible involvement of MAPs or MAP-like proteins in DNA replication, in vivo.     

Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans

Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-->5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-->5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase.     

Inhibition of DNA polymerase alpha activity by proteins from rat liver

We determined that there is a protein in rat liver capable of inhibiting DNA polymerase alpha. To assay for this inhibitor, DNA polymerase alpha was purified from R3230AC rat mammary tumor, a rich source of this enzyme. Protein fractions from Sephacryl S200 gel filtration of total soluble liver extract showing inhibition of DNA polymerase alpha were further chromatographed on DEAE-cellulose. This step revealed two inhibitor protein populations with the major form corresponding to a molecular weight of 143,000 dalton. Soluble extract from isolated rat liver nuclei also showed the presence of at least two inhibitors; the major form was 200,000+ dalton in molecular weight. Both the 143,000 and 200,000+ dalton inhibitor proteins were capable of inhibiting the R3230AC tumor DNA polymerase alpha in a dose-dependent manner. These inhibitors exhibited similar inhibition of nuclear matrix-associated DNA polymerase alpha from either the R3230AC tumor or from regenerating rat liver.     

Elongation of repetitive DNA by DNA polymerase from a hyperthermophilic bacterium Thermus thermophilus

Short repetitive DNA sequences are believed to be one of the primordial genetic elements that served as a source of complex large DNA found in the genome of modern organisms. However, the mechanism of its expansion (increase in repeat number) during the course of evolution is unclear. We demonstrate that the DNA polymerase of the hyperthermophilic bacterium Thermus thermophilus can elongate oligoDNA with several tandem repeats to very long DNA in vitro. For instance, 48mer repetitive oligoDNA (TACATGTA)₆, which has 25% GC content and a palindromic sequence, can be elongated up to ~10 000 bases by DNA polymerase at 74°C without template DNA. OligoDNA having a different GC content or a quasi-palindromic sequence can also be elongated, but less efficiently. A spectroscopic thermal melting experiment with the oligoDNA showed that its hairpin–coil transition temperature was very close to the elongation reaction temperature (74°C), but was much higher than the temperature at which duplex oligoDNA can exist stably. Taken together, we conclude that repetitive oligoDNA with a palindromic or quasi-palindromic sequence is elongated extensively by a hyperthermophilic DNA polymerase through hairpin–coil transitions. We propose that such an elongation mechanism might have been a driving force to expand primordial short DNA.     

Influence of chromatin and single strand binding proteins on the activity of an archaeal MCM

The mini-chromosome maintenance (MCM) complex is the presumptive replicative helicase in archaea and eukaryotes. In archaea, the MCM is a homo-multimer, in eukaryotes a heterohexamer composed of six related subunits, MCM 2-7. Biochemical studies using naked DNA templates have revealed that archaeal MCMs and a sub-complex of eukaryotic MCM 4, 6 and 7 have 3' to 5' helicase activity. Here, we investigate the influence of the major chromatin proteins, Alba and Sul7d, of Sulfolobus solfataricus (Sso) on the ability of the MCM complex to melt partial duplex DNA substrates. In addition, we test the effect of Sso SSB on MCM activity. We reveal that Alba represents a formidable barrier to MCM activity and further demonstrate that acetylation of Alba alleviates repression of MCM activity.     

Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

An interesting property of the Escherichia coli DNA polymerase II is the stimulation in DNA synthesis mediated by the DNA polymerase III accessory proteins beta,gamma complex. In this paper we have studied the basis for the stimulation in pol II activity and have concluded that these accessory proteins stimulate pol II activity by increasing the processivity of the enzyme between 150- and 600-fold. As is the case with pol III, processive synthesis by pol II requires both beta,gamma complex and SSB protein. Whereas the intrinsic velocity of synthesis by pol II is 20-30 nucleotides per s with or without the accessory proteins, the processivity of pol II is increased from approximately five nucleotides to greater than 1600 nucleotides incorporated per template binding event. The effect of the accessory proteins on the rate of replication is far greater on pol III than on pol II; pol III holoenzyme is able to complete replication of circular single-stranded M13 DNA in less than 20 s, whereas pol II in the presence of the gamma complex and beta requires approximately 5 min. We have investigated the effect of beta,gamma complex proteins on bypass of a site-specific abasic lesion by E. coli DNA polymerases I, II, and III. All three polymerases are extremely inefficient at bypass of the abasic lesion. We find limited bypass by pol I with no change upon addition of accessory proteins. pol II also shows limited bypass of the abasic site, dependent on the presence of beta,gamma complex and SSB. pol III shows no significant bypass of the abasic site with or without beta,gamma complex.     

Modulation of DNA end joining by nuclear proteins

DNA double strand breaks in mammalian cells are primarily repaired by homologous recombination and non-homologous end joining (NHEJ). NHEJ may either be error-free or mutagenic with deletions or insertions at the joint. Recent studies showed that DNA ends can also be joined via microhomologous sequences flanking the break point especially when proteins responsible for NHEJ, such as Ku, are absent. Microhomology-mediated end joining (MHEJ) is always accompanied by a deletion that spans one of the two homologous sequences and the intervening sequence, if any. In this study we evaluated several factors affecting the relative contribution of MHEJ to DNA end joining using nuclear extracts and DNA substrates containing 10-bp repeats at the ends. We found that the occurrence of MHEJ is determined by the relative abundance of nuclear proteins. At low DNA/protein ratios, an error-free end-joining mechanism predominated over MHEJ. As the DNA/protein ratio increased, MHEJ became predominant. We show that the nuclear proteins that contribute to the inhibition of the error-prone MHEJ include Ku and histone H1. Treatment of extracts with flap endonuclease 1 antiserum significantly reduced MHEJ. Addition of a 17-bp intervening sequence between the microhomologous sequences significantly reduced the efficiency of MHEJ. Thus, this cell-free assay provides a platform for evaluating factors modulating end joining.     

Effects of nuclear proteins on the activity of soybean DNA polymerase

The effects of nuclear proteins on DNA synthesis were investigated before and after incubation with radioactive ATP and a crude preparation of nuclear protein kinase. After partial purification by DEAE-cellulose chromatography, the major protein fractions were added separately to DNA polymerase assays. One of the seven protein fractions inhibited DNA synthesis by 50%, whereas three other fractions stimulated DNA polymerase activity 3 to 4-fold. After incubation with ATP, one fraction became inhibitory, and the three stimulatory fractions, which had high levels of radioactivity, were more effective. This stimulation of DNA polymerase activity was proportional to added nuclear protein and was maximum at $\text{6 μ}\text{g}\text{20 μ}\text{g}$ DNA.     

Efficient and high fidelity incorporation of dye-terminators by a novel archaeal DNA polymerase mutant

We examined the molecular basis of ddNTP selectivity in archaeal family B DNA polymerases by randomly mutagenizing the gene encoding Thermococcus sp. JDF-3 DNA polymerase and screening mutant libraries for improved ddNTP incorporation. We identified two mutations, P410L and A485T, that improved ddNTP uptake, suggesting the contribution of P410 and A485 to ddNTP/dNTP selectivity in archaeal DNA polymerases. The importance of A485 was identified previously in mutagenesis studies employing Pfu (A486) and Vent (A488) DNA polymerases, while the contribution of P410 to ddNTP/dNTP selectivity has not been reported. We demonstrate that a combination of mutations (P410L/A485T) has an additive effect in improving ddNTP incorporation by a total of 250-fold. To assess the usefulness of the JDF-3 P410L/A485T in fluorescent-sequencing applications, we compared the archaeal mutant to Taq F667Y with respect to fidelity and kinetic parameters for DNA and dye-ddNTPs. Although the Taq F667Y and JDF-3 P410L/A485T mutants exhibit similar K(m) and V(max) values for dye-ddNTPs in single-base extension assays, the archaeal mutant exhibits higher fidelity due to a reduced tendency to form certain (ddG:dT, ddT:dC) mispairs. DNA polymerases exhibiting higher insertion fidelity are expected to provide greater accuracy in SNP frequency determinations by single-base extension and in multiplex minisequencing assays.     

Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: a mechanism of DNA elongation and diversification

DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.