Replicative DNA Polymerases

In 1959, Arthur Kornberg was awarded the Nobel Prize for his work on the principles by which DNA is duplicated by DNA polymerases. Since then, it has been confirmed in all branches of life that replicative DNA polymerases require a single-stranded template to build a complementary strand, but they cannot start a new DNA strand de novo. Thus, they also depend on a primase, which generally assembles a short RNA primer to provide a 3′-OH that can be extended by the replicative DNA polymerase. The general principles that (1) a helicase unwinds the double-stranded DNA, (2) single-stranded DNA-binding proteins stabilize the single-stranded DNA, (3) a primase builds a short RNA primer, and (4) a clamp loader loads a clamp to (5) facilitate the loading and processivity of the replicative polymerase, are well conserved among all species. Replication of the genome is remarkably robust and is performed with high fidelity even in extreme environments. Work over the last decade or so has confirmed (6) that a common two-metal ion-promoted mechanism exists for the nucleotidyltransferase reaction that builds DNA strands, and (7) that the replicative DNA polymerases always act as a key component of larger multiprotein assemblies, termed replisomes. Furthermore (8), the integrity of replisomes is maintained by multiple protein–protein and protein–DNA interactions, many of which are inherently weak. This enables large conformational changes to occur without dissociation of replisome components, and also means that in general replisomes cannot be isolated intact.The genomes, from the smallest to the largest, provide an enormous challenge for the replicative DNA polymerases to faithfully copy to give the many generations that follow a comparable condition for life. In this article, we discuss the structural and functional bases by which replicative DNA polymerases are able to efficiently and faithfully build new copies of genomes in eubacteria, archaea, and eukaryotes.     

DNA Polymerases from Human Cells

Nuclei of HeLa cells contain two separable DNA polymerases which have different properties and cytoplasm contains a single DNA polymerase activity similar to one of the nuclear DNA polymerases. The same distribution of DNA polymerases has been found in the nucleus and cytoplasm of WI-38 cells, a normal diploid human cell line.     

Repair of DNA lesions: mechanisms and relative repair efficiencies

DNA is frequently damaged by endogenous agents inside the cells. Some exogenous agents such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and may thus contribute to the 'background' DNA damage in humans. DNA lesions are normally removed by various repair mechanisms. The major repair mechanisms for various DNA lesions are summarized. In contrast to the extensively studied repair mechanisms, much less is known about the relative repair efficiencies of various DNA lesions. Since DNA repair is a crucial defense against carcinogenesis, it may constitute an important factor affecting the carcinogenicity of DNA damaging agents. We have adopted a human cell-free system for measuring relative DNA repair efficiencies based on the concept of repair competition between acetylaminofluorene adducts and other DNA lesions of interest. Using this in vitro system, we determined the relative repair efficiencies of PAH adducts induced by: anti-(+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), anti-(+/-)-benz[a]anthracene-trans-3,4-dihydrodiol-1,2-epoxide (BADE-I), anti-(+/-)-benz[a]anthracene-trans-8,9-dihydrodiol-10, 11-epoxide (BADE-II), anti-(+/-)-benzo[b]fluoranthene-trans-9, 10-dihydrodiol-11,12-epoxide (BFDE), anti-(+/-)-chrysene-trans-1, 2-dihydrodiol-3,4-epoxide (CDE), and anti-(+/-)-dibenzo[a, l]pyrene-trans-11,12-dihydrodiol-13,14-epoxide (DBPDE). While damage by BPDE, DBPDE, CDE, and BFDE were repaired by nucleotide excision repair as efficiently as AAF adducts, the repair of BADE-I and BADE-II adducts were significantly slower in human cell extracts. Damage by DBPDE at 3 microM in vitro yielded approximately 5-fold higher DNA adducts than BPDE as determined by quantitative PCR. This potent DNA reactivity may account in part for the potent carcinogenicity of dibenzo[a,l]pyrene. The correlation of these results to the carcinogenic properties of the PAH compounds is discussed. Furthermore, we show that NER plays a role in AP site repair in vivo in the eukaryotic model organism yeast.     

DNA Polymerases that Propagate the Eukaryotic DNA Replication Fork

Abstract

Three DNA polymerases are thought to function at the eukaryotic DNA replication fork. Currently, a coherent model has been derived for the composition and activities of the lagging strand machinery. RNA-DNA primers are initiated by DNA polymerase α -primase. Loading of the proliferating cell nuclear antigen, PCNA, dissociates DNA polymerase α and recruits DNA polymerase δ and the flap endonuclease FEN1 for elongation and in preparation for its requirement during maturation, respectively. Nick translation by the strand displacement action of DNA polymerase δ, coupled with the nuclease action of FEN1, results in processive RNA degradation until a proper DNA nick is reached for closure by DNA ligase I. In the event of excessive strand displacement synthesis, other factors, such as the Dna2 nuclease/helicase, are required to trim excess flaps. Paradoxically, the composition and activity of the much simpler leading strand machinery has not been clearly established. The burden of evidence suggests that DNA polymerase ε normally replicates this strand, but under conditions of dysfunction, DNA polymerase δ may substitute.     

SOS-Inducible DNA Polymerases and Adaptive Mutagenesis

Stability of genomes of living organisms is maintained by various mechanisms that ensure high fidelity of DNA replication. However, cells can reversibly enhance the level of replication errors in response to external factors. As mutable states are potentially involved in carcinogenesis, aging, and resistance for pathogenic agents, the existence of these states is of great importance for human health. A well-known system of inducible mutation is SOS response, whose key component is replication of damaged DNA regions. Inducible mutation implies a contribution of SOS response to the adaptation of a bacterial population to adverse environments. There is ample evidence indicating the primary role of SOS response genes in the phenomenon of adaptive mutation. The involvement of the SOS system in adaptive mutagenesis is discussed.     

Base Excision Repair: AP Endonucleases and DNA Polymerases

The DNA base lesions in living cells occur permanently and with high frequency as a result of the action of exogenous and endogenous factors. The main mechanism providing removal of such lesions is base excision repair.     

RecA-mediated excision repair: a novel mechanism for repairing DNA lesions at sites of arrested DNA synthesis

In Escherichia coli, bulky DNA lesions are repaired primarily by nucleotide excision repair (NER). Unrepaired lesions encountered by DNA polymerase at the replication fork create a blockage which may be relieved through RecF-dependent recombination. We have designed an assay to monitor the different mechanisms through which a DNA polymerase blocked by a single AAF lesion may be rescued by homologous double-stranded DNA sequences. Monomodified single-stranded plasmids exhibit low survival in non-SOS induced E. coli cells; we show here that the presence of a homologous sequence enhances the survival of the damaged plasmid more than 10-fold in a RecA-dependent way. Remarkably, in an NER proficient strain, 80% of the surviving colonies result from the UvrA-dependent repair of the AAF lesion in a mechanism absolutely requiring RecA and RecF activity, while the remaining 20% of the surviving colonies result from homologous recombination mechanisms. These results uncover a novel mechanism - RecA-mediated excision repair - in which RecA-dependent pairing of the mono-modified single-stranded template with a complementary sequence allows its repair by the UvrABC excinuclease.     

Surviving the sun: repair and bypass of DNA UV lesions

Structural studies of UV-induced lesions and their complexes with repair proteins reveal an intrinsic flexibility of DNA at lesion sites. Reduced DNA rigidity stems primarily from the loss of base stacking, which may manifest as bending, unwinding, base unstacking, or flipping out. The intrinsic flexibility at UV lesions allows efficient initial lesion recognition within a pool of millions to billions of normal DNA base pairs. To bypass the damaged site by translesion synthesis, the specialized DNA polymerase η acts like a molecular "splint" and reinforces B-form DNA by numerous protein-phosphate interactions. Photolyases and glycosylases that specifically repair UV lesions interact directly with UV lesions in bent DNA via surface complementation. UvrA and UvrB, which recognize a variety of lesions in the bacterial nucleotide excision repair pathway, appear to exploit hysteresis exhibited by DNA lesions and conduct an ATP-dependent stress test to distort and separate DNA strands. Similar stress tests are likely conducted in eukaryotic nucleotide excision repair.     

DNA repair is responsible for the presence of oxidatively damaged DNA lesions in urine

The repair of oxidatively damaged DNA is integral to the maintenance of genomic stability, and hence prevention of a wide variety of pathological conditions, such as aging, cancer and cardiovascular disease. The ability to non-invasively assess DNA repair may provide information regarding repair pathways, variability in repair capacity, and susceptibility to disease. The development of assays to measure urinary DNA lesions offered this potential, although it rapidly became clear that possible contribution from diet and cell turnover may influence urinary lesion levels. Whilst early studies attempted to address these issues, up until now, much of the data appears conflicting. However, recent work from our laboratories, in which human volunteers were fed highly oxidatively modified 15N-labelled DNA demonstrates that diet does not appear to contribute to urinary levels of 8-hydroxyguanine and 7,8-dihydro-8-oxo-2'-deoxyguanosine. Furthermore, we propose that a number of literature reports form an argument against a contribution from cell death. Indeed we, and others, have presented evidence, which strongly suggests the involvement of cell death to be minimal. Taken together, these data would appear to rule out various confounding factors, leaving DNA repair pathways as the principal source of urinary purine, if not DNA, lesions enabling such measurements to be used as indicators of repair.     

Light and dark in chromatin repair: repair of UV-induced DNA lesions by photolyase and nucleotide excision repair

Nucleotide excision repair (NER) and DNA repair by photolyase in the presence of light (photoreactivation) are the major pathways to remove UV-induced DNA lesions from the genome, thereby preventing mutagenesis and cell death. Photoreactivation was found in many prokaryotic and eukaryotic organisms, but not in mammals, while NER seems to be universally distributed. Since packaging of eukaryotic DNA in nucleosomes and higher order chromatin structures affects DNA structure and accessibility, damage formation and repair are coupled intimately to structural and dynamic properties of chromatin. Here, I review recent progress in the study of repair of chromatin and transcribed genes. Photoreactivation and NER are discussed as examples of how an individual enzyme and a complex repair pathway, respectively, access DNA lesions in chromatin and how these two repair processes fulfil complementary roles in removal of UV lesions. These repair pathways provide insight into the structural and dynamic properties of chromatin and suggest how other DNA repair processes could work in chromatin.     

Synthesis and Recognition by DNA Polymerases of a Reactive Nucleoside for DNA Diversification

Abstract

The synthesis of 1-(2-deoxy-β-D-erythro-pentofuranosyl)imidazole-4-hydrazide having the features of an ambigous base is reported. The recognition of the analogue by DNA polymerases as an incoming triphosphate as well as a template base was investigated. The mutagenic properties was evaluated by PCR. The potential of this new monomer for DNA diversification is illustrated by the reactivity of the nucleobase towards various aldehydes.     

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.     

When DNA repair goes wrong: BER-generated DNA-protein crosslinks to oxidative lesions

Free radicals generate an array of DNA lesions affecting all parts of the molecule. The damage to deoxyribose receives less attention than base damage, even though the former accounts for ∼20% of the total. Oxidative deoxyribose fragments (e.g., 3′-phosphoglycolate esters) are removed by the Ape1 AP endonuclease and other enzymes in mammalian cells to enable DNA repair synthesis. Oxidized abasic sites are initially incised by Ape1, thus recruiting these lesions into base excision repair (BER) pathways. Lesions such as 2-deoxypentos-4-ulose can be removed by conventional (single-nucleotide) BER, which proceeds through a covalent Schiff base intermediate with DNA polymerase β (Polβ) that is resolved by hydrolysis. In contrast, the lesion 2-deoxyribonolactone (dL) must be processed by multinucleotide (“long-patch”) BER: attempted repair via the single-nucleotide pathway leads to a dead-end, covalent complex with Polβ cross- linked to the DNA by an amide bond. We recently detected these stable DNA-protein crosslinks (DPC) between Polβ and dL in intact cells. The features of the DPC formation in vivo are exactly in keeping with the mechanistic properties seen in vitro: Polβ-DPC are formed by oxidative agents in line with their ability to form the dL lesion; they are not formed by non-oxidative agents; DPC formation absolutely requires the active-site lysine-72 that attacks the 5′-deoxyribose; and DPC formation depends on Ape1 to incise the dL lesion first. The Polβ-DPC are rapidly processed in vivo, the signal disappearing with a half-life of 15–30 min in both mouse and human cells. This removal is blocked by inhibiting the proteasome, which leads to the accumulation of ubiquitin associated with the Polβ-DPC. While other proteins (e.g., topoisomerases) also form DPC under these conditions, 60–70% of the trapped ubiquitin depends on Polβ. The mechanism of ubiquitin targeting to Polβ-DPC, the subsequent processing of the expected 5′-peptidyl-dL, and the biological consequences of unrepaired DPC are important to assess. Many other lyase enzymes that attack dL can also be trapped in DPC, so the processing mechanisms may apply quite broadly.     

Mammalian polymerase ζ is essential for post-replication repair of UV-induced DNA lesions

DNA polymerase ζ is believed to be an essential constituent of DNA damage tolerance, comprising several pathways that allow the replication of DNA templates containing unrepaired damage. We wanted to better define the role of polymerase ζ in DNA damage tolerance in mammalian cells. To this aim we have investigated replication of ultraviolet light-damaged DNA templates in mouse embryonic fibroblasts deficient for Rev3, the catalytic subunit of polymerase ζ. We found that Rev3 is important for a post-replication repair pathway of helix-distorting [6-4]pyrimidine-pyrimidone photoproducts and, to a lesser extent, of cyclobutane pyrimidine dimers. Unlike its partner Rev1, Rev3 appears not to be involved in an immediate translesion synthesis pathway at a stalled replication fork. The deficiency of Rev3^?/? MEFs in post-replication repair of different photoproducts contributes to the extreme sensitivity of these cells to UV light.     

Deficient repair of DNA lesions in Alzheimer's disease fibroblasts

DNA strand breaks, resulting from treatment with N-methyl-N'-nitro-N-nitrosoguanidine, were repaired more slowly in four strains of familial Alzheimer's disease fibroblasts than in five strains of fibroblasts from age-matched normals. These results were not due to differences between the two cell types in in vitro ages, in the initial DNA damage or in drug-induced cell lysis. Bleomycin-induced DNA double-strand breaks were repaired equally efficiently by both types of cells. Alzheimer's disease cells may have a DNA repair defect, which may be involved in the pathogenesis of this disease.     

Selective Inhibition of DNA Chain Elongation Catalyzed by DNA Polymerases

Abstract

The results of series of works on the properties of a large number of nucleoside 5′-triphosphates analogs in the reaction catalyzed by several DNA polymerases are summarized.

Molecular mechanisms of substrate selection by DNA polymerases are not studied in detail. Therefore we have undertaken a comparative analysis of DNA polymerases from different sources employing nucleoside 5′-triphosphate analogs capable of incorporating into DNA chains terminating these chains elongation. Synthesis of a large line of nucleoside 5′-triphosphate analogs with substitution at the sugar residue has been performed. DNA polymerases have been isolated, and the synthesis of DNA has been studied using phage M13 DNA or phage MS2 RNA with synthetic deoxyoligonucleoti-de primers. The molecular mechanism of the substrate action has been determined by PAG electrophoresis of the reaction