Translesion synthesis: Y-family polymerases and the polymerase switch

Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes mono-ubiquitination of PCNA plays a crucial role in the switch from replicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks.     

Deoxyribonucleic acid-dependent ribonucleic acid polymerases from normal and polyoma-transformed BHK-21/C13 cells.

DNA-dependent RNA polymerase (EC 2.7.7.6) ACTIVITIES FROM NORMAL BHK-21/C13 cells and from BHK-21/C13 cells transformed by polyoma virus (PYY cells) were solubilized and fractionated on columns of DEAE-Sephadex. Various properties of the A and B enzymes from the two types of cell were compared. 1. The yields of polymerase relative to the DNA content of the nuclear preparations are similar for both cell types. 2. The ionic-strength optima of polymerases A and B are 12.5 mM and 100mM with respect to (NH4)2SO4 for both cell types. 3. The Mn2+/Mg2+ activity ratio (measured at the respective optimum for each cation) for polymerase A from BHK-21/C13 cells was 1.48 and for the polymerase A from PYY cells was 0.55. The corresponding ratios for polymerase B were 10.11 for BHK-21/C13 cells and 22.75 for PYY cells. 4. Minor differences in the ability of the A polymerases to transcribe native and denatured DNA templates were observed; such differences were not apparent when the B polymerases were compared. 5. All the polymerases were inhibited completely by actinomycin D and by rifampicin AF/013, but not markedly so by rifampicin. Alpha-amanitin inhibited polymerase B but not polymerase A.     

phi 29 DNA polymerase residue Leu384, highly conserved in motif B of eukaryotic type DNA replicases,is involved in nucleotide insertion fidelity

Replicative DNA polymerases achieve insertion fidelity by geometric selection of a complementary nucleotide followed by induced fit: movement of the fingers subdomain toward the active site to enclose the incoming and templating nucleotides generating a binding pocket for the nascent base pair. Several residues of motif B of DNA polymerases from families A and B, localized in the fingers subdomain, have been described to be involved in template/primer binding and dNTP selection. Here we complete the analysis of this motif, which has the consensus "KLX2NSXYG" in DNA polymerases from family B, characterized by mutational analysis of conserved leucine, Leu384 of phi 29 DNA polymerase. Mutation of Leu384 into Arg resulted in a phi 29 DNA polymerase with reduced nucleotide insertion fidelity during DNA-primed polymerization and protein-primed initiation reactions. However, the mutation did not alter the intrinsic affinity for the different dNTPs, as shown in the template-independent terminal protein-deoxynucleotidylation reaction. We conclude that Leu384 of phi 29 DNA polymerase plays an important role in positioning the templating nucleotide at the polymerization active site and in controlling nucleotide insertion fidelity. This agrees with the localization of the corresponding residue in the closed ternary complexes of family A and family B DNA polymerases, contributing to form the binding pocket for the nascent base pair. As an additional effect, mutant polymerase L384R was strongly reduced in DNA binding, resulting in reduced processivity during polymerization.     

Structural and biochemical investigation of the role in proofreading of a beta hairpin loop found in the exonuclease domain of a replicative DNA polymerase of the B family

Replicative DNA polymerases, as exemplified by the B family polymerases from bacteriophages T4 and RB69, not only replicate DNA but also have the ability to proofread misincorporated nucleotides. Because the two activities reside in separate protein domains, polymerases must employ a mechanism that allows for efficient switching of the primer strand between the two active sites to achieve fast and accurate replication. Prior mutational and structural studies suggested that a beta hairpin structure located in the exonuclease domain of family B polymerases might play an important role in active site switching in the event of a nucleotide misincorporation. We show that deleting the beta hairpin loop in RB69 gp43 affects neither polymerase nor exonuclease activities. Single binding event studies with mismatched primer termini, however, show that the beta hairpin plays a role in maintaining the stability of the polymerase/DNA interactions during the binding of the primer DNA in the exonuclease active site but not on the return of the corrected primer to the polymerase active site. In addition, the deletion variant showed a more stable incorporation of a nucleotide opposite an abasic site. Moreover, in the 2.4 A crystal structure of the beta hairpin deletion variant incorporating an A opposite a templating furan, all four molecules in the crystal asymmetric unit have DNA in the polymerase active site, despite the presence of DNA distortions because of the misincorporation, confirming that the primer strand is not stably bound within the exonuclease active site in the absence of the beta hairpin loop.     

Xeroderma pigmentosum variant and error-prone DNA polymerases

Replicative DNA synthesis is a faithful event which requires undamaged DNA and high fidelity DNA polymerases. If unrepaired damage remains in the template DNA during replication, specialised low fidelity DNA polymerases synthesises DNA past lesions (translesion synthesis, TLS). Current evidence suggests that the polymerase switch from replicative to translesion polymerases might be mediated by post-translational modifications involving ubiquitination processes. One of these TLS polymerases, polymerase eta carries out TLS past UV photoproducts and is deficient in the variant form of xeroderma pigmentosum (XP-V). The dramatic proneness to skin cancer of XP-V individuals highlights the importance of this DNA polymerase in cancer avoidance. The UV hypermutability of XP-V cells suggests that, in the absence of a functional poleta, UV-induced lesions are bypassed by inaccurate DNA polymerase(s) which remain to be identified.     

Levels of DNA polymerases alpha, beta, and gamma in control and repair-deficient human diploid fibroblasts 1.

The activities of DNA polymerases alpha, beta, and gamma were determined in control and repair-deficient human fibroblasts (xeroderma pigmentosum complementation groups A, C, and D; Fanconi's Anemia; and Bloom's syndrome). Assays were done on 103,000XG supernatants which had been chromatographed on DEAE cellulose to remove nucleic acids and on fractions containing polymerase activities which had been separated from one another on a second DEAE cellulose column. All repair-deficient cell types contained all three DNA polymerase activities. Caffeine, which has been observed to inhibit some DNA-repair processes in intact cells, had no effect on DNA polymerase activities from XP-A, XP-C, XP-D or XP-variant cells. These data indicate that all three polymerases are present in cells which have reduced or absent repair functions and that the caffeine effects observed in living cells are probably not due to the direct action of caffeine on DNA polymerases.     

DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities.

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.     

Mammalian proliferating cell nuclear antigen stimulates the processivity of two wheat embryo DNA polymerases.

Multiple DNA polymerases have been described in all organisms studied to date. Their specific functions are not easy to determine, except when powerful genetic and/or biochemical tools are available. However, the processivity of a DNA polymerase could reflect the physiological role of the enzyme. In this study, analogies between plant and animal DNA polymerases have been investigated by analyzing the size of the products synthesized by wheat DNA polymerases A, B, CI, and CII as a measure of their processivity. Thus, incubations have been carried out with poly(dA)-oligo(dT) as a template-primer under varying assay conditions. In the presence of MgCl2, DNA polymerase A was highly processive, whereas DNA polymerases B, CI, and CII synthesized much shorter products. With MnCl2 instead of MgCl2, DNA polymerase A was highly processive, DNA polymerases B and CII were moderately processive, and DNA polymerase CI remained strictly distributive. The effect of calf thymus proliferating cell nuclear antigen (PCNA) on wheat polymerases was studied as described for animal DNA polymerases. The high processivity of DNA polymerase A was PCNA independent, whereas both enzyme activity and processivity of wheat DNA polymerases B and CII were significantly stimulated by PCNA. On the other hand, DNA polymerase CI was not stimulated by PCNA and, like animal DNA polymerase beta, was distributive in all cases. From these results, we propose that wheat DNA polymerase A could correspond to a DNA polymerase alpha, DNA polymerases B and CII could correspond to the delta-like enzyme, and DNA polymerase CI could correspond to DNA polymerase beta.     

Evolution of DNA Polymerase Families: Evidences for Multiple Gene Exchange Between Cellular and Viral Proteins

A phylogenetic analysis of the five major families of DNA polymerase is presented. Viral and plasmid sequences are included in this compilation along with cellular enzymes. The classification by Ito and Braithwaite (Ito and Braithwaite 1991) of the A, B, C, D, and X families has been extended to accommodate the ``Y family' of DNA polymerases that are related to the eukaryotic RAD30 and the bacterial UmuC gene products. After analysis, our data suggest that no DNA polymerase family was universally conserved among the three biological domains and no simple evolutionary scenario could explain that observation. Furthermore, viruses and plasmids carry a remarkably diverse set of DNA polymerase genes, suggesting that lateral gene transfer is frequent and includes non-orthologous gene displacements between cells and viruses. The relationships between viral and host genes appear very complex. We propose that the gamma DNA polymerase of the mitochondrion replication apparatus is of phage origin and that this gene replaced the one in the bacterial ancestor. Often there was no obvious relation between the viral and the host DNA polymerase, but an interesting exception concerned the family B enzymes: in which ancient gene exchange can be detected between the viruses and their hosts. Additional evidence for horizontal gene transfers between cells and viruses comes from an analysis of the small damage-inducible DNA polymerases. Taken together, these findings suggest a complex evolutionary history of the DNA replication apparatus that involved significant exchanges between viruses, plasmids, and their hosts.     

The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Subcellular distribution, exonuclease activity and heterogeneity of the enzymes.

Four DNA polymerases from the marine diatom Cylindrotheca fusiformis, polymerases A, B, C and D, were further differentiated by their subcellular localization, presence of deoxyribonuclease activity, apparent heterogeneity and molecular weights. Polymerases A, B and D occur in significant amounts in the soluble fraction, suggesting that they were originally localized in the nuclei, whereas polymerase C predominates in the chloroplasts. A mitochondrial DNA polymerase was also isolated and characterized by ion-exchange chromatography. Polymerase D has an associated nuclease activity which prefers denatured DNA and Mg2+, and has a pH optimum higher than that for polymerase activity. Co-elution from a DEAE-Sephadex column and co-sedimentation in glycerol density gradients of deoxyribonuclease and polymerase D activity suggest a molecular association. Polymerases A, B and C are devoid of nuclease activity. Glycerol-gradient-sedimentation analysis showed that all DNA polymerase fractions are heterogeneous at low ionic strengths, with the appearance of a single homogeneous activity of 0.5M-KCl. Estimated molecular weights of 100000, 82000 and 120000 for polymerases A, B and C respectively were obtained from sedimentation analysis and gel filtration. Polymerase D was estimated to have a molecular weight of about 100000 as determined by sedimentation analysis alone.     

Characterization of a triple DNA polymerase replisome

The replicase of all cells is thought to utilize two DNA polymerases for coordinated synthesis of leading and lagging strands. The DNA polymerases are held to DNA by circular sliding clamps. We demonstrate here that the E. coli DNA polymerase III holoenzyme assembles into a particle that contains three DNA polymerases. The three polymerases appear capable of simultaneous activity. Furthermore, the trimeric replicase is fully functional at a replication fork with helicase, primase, and sliding clamps; it produces slightly shorter Okazaki fragments than replisomes containing two DNA polymerases. We propose that two polymerases can function on the lagging strand and that the third DNA polymerase can act as a reserve enzyme to overcome certain types of obstacles to the replication fork.     

DNA polymerases in midgestation mouse embryo, trophoblast, and decidua

The DNA polymerases of midgestation mouse embryo, trophoblast, and decidua have been examined. A low molecular weight, nuclear. DNA-dependent polymerase (D-DNA polymerase) and a higher molecular weight cytoplasmic enzyme were found in all three cell types. A DNA polymerase which utilized the poly(A) strand of oligo(dT) · poly(A) as template (R-DNA polymerase) was also found in the three cell types. This enzyme was present both in the nucleus and the cytoplasm. All enzyme levels were highest in the rapidly dividing embryonic cells, substantially lower in the DNA replicating but nondividing trophoblast cells, and lowest in the nonreplicating, nondividing decidual cells. Our observations are consistent with the idea that the nuclear and cytoplasmic D-DNA polymerases are under coordinate control. The relationship of these enzymes to DNA synthesis in vivo is discussed.     

An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site

Substrate-induced conformational change of the protein is the linchpin of enzymatic reactions. Replicative DNA polymerases, for example, convert from an open to a closed conformation in response to dNTP binding. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, differs strikingly from other polymerases in its much higher proficiency and fidelity for nucleotide incorporation opposite template purines than opposite template pyrimidines. We present here a crystallographic analysis of hPoliota binary complexes, which together with the ternary complexes show that, contrary to replicative DNA polymerases, the DNA, and not the polymerase, undergoes the primary substrate-induced conformational change. The incoming dNTP "pushes" templates A and G from the anti to the syn conformation dictated by a rigid hPoliota active site. Together, the structures posit a mechanism for template selection wherein dNTP binding induces a conformational switch in template purines for productive Hoogsteen base pairing.     

Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures ~100°C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases δ and ε for nuclear DNA and polymerase γ for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.     

DNA replication fidelity

DNA replication fidelity plays fundamental role in faithful transmission of genetic material during cell division and during transfer of genetic material from parents to progeny. Replicative polymerases are the main guardian responsible for high replication fidelity of genomic DNA. DNA main replicative polymerases are also involved in many DNA repair processes. High fidelity of DNA replication is determined by correct nucleotide selectivity in polymerase active center, and exonucleolytic proofreading that removes mismatches from primer terminus. In this article we will focus on the mechanisms that are responsible for high fidelity of replications with the special emphasis on structural studies showing important conformational changes after substrate binding. We will also stress the importance of hydrogen bonding, base pair geometry, polymerase DNA interactions and the role of accessory proteins in replication fidelity.     

Sequence analysis of three family B DNA polymerases from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis.

Three family B DNA polymerase genes, designated B1, B2, and B3, were cloned from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis, and sequenced. Deduced amino acid sequences of B1 and B3 DNA polymerases have all exonuclease and polymerase motifs which include critical residues for catalytic activities. Furthermore, a YxGG/A motif, which is located between 3'-5' exonuclease and polymerization domains of family B DNA polymerases, was also found in each of the B1 and B3 sequences. These findings suggested that S. ohwakuensis B1 and B3 DNA polymerases have both exonuclease and polymerase activities. However, amino acid sequence of the B2 DNA polymerase of this organism contains several amino acid substitutions in Pol-motifs, and also lacks Exo-motif I and Exo-motif II. These substitutions and lack of certain motifs raise questions about polymerase and exonuclease activities of the corresponding gene product. The B3 sequence of S. ohwakuensis is more closely related to Pyrodictium, Aeropyrum, and Archaeoglobus DNA polymerase B3 sequences than to the Sulfolobus B3 sequences. Phylogenetic analysis showed that crenarchaeal B1 DNA polymerases are closely related to each other, and suggested that crenarchaeal B3, euryarchaeal family B, and eukaryal epsilon DNA polymerases may be orthologs.