DNA polymerases and human diseases

DNA polymerases function in DNA replication, repair, recombination and translesion synthesis. Currently, 15 DNA polymerase genes have been identified in human cells, belonging to four distinct families. In this review, we briefly describe the biochemical activities and known cellular roles of each DNA polymerase. Our major focus is on the phenotypic consequences of mutation or ablation of individual DNA polymerase genes. We discuss phenotypes of current mouse models and altered polymerase functions and the relationship of DNA polymerase gene mutations to human cell phenotypes. Interestingly, over 120 single nucleotide polymorphisms (SNPs) have been identified in human populations that are predicted to result in nonsynonymous amino acid substitutions of DNA polymerases. We discuss the putative functional consequences of these SNPs in relation to human disease.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

Distinctive activities of DNA polymerases during human DNA replication

The contributions of human DNA polymerases (pols) alpha, delta and epsilon during S-phase progression were studied in order to elaborate how these enzymes co-ordinate their functions during nuclear DNA replication. Pol delta was three to four times more intensely UV cross-linked to nascent DNA in late compared with early S phase, whereas the cross-linking of pols alpha and epsilon remained nearly constant throughout the S phase. Consistently, the chromatin-bound fraction of pol delta, unlike pols alpha and epsilon, increased in the late S phase. Moreover, pol delta neutralizing antibodies inhibited replicative DNA synthesis most efficiently in late S-phase nuclei, whereas antibodies against pol epsilon were most potent in early S phase. Ultrastructural localization of the pols by immuno-electron microscopy revealed pol epsilon to localize predominantly to ring-shaped clusters at electron-dense regions of the nucleus, whereas pol delta was mainly dispersed on fibrous structures. Pol alpha and proliferating cell nuclear antigen displayed partial colocalization with pol delta and epsilon, despite the very limited colocalization of the latter two pols. These data are consistent with models where pols delta and epsilon pursue their functions at least partly independently during DNA replication.     

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase gamma type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called 'cytoplasmic' DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase alpha from normal cells: DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase beta.     

Structural and functional relationships of human DNA polymerases

A continuing theme of our laboraory, has been the understanding of human DNA polymerases at the structural level. We have purified DNA polymerases delta, epsilon and alpha from human placenta. Monoclonal antibodies to these polymerases were isolated and used as tools to study their immunochemical relationships. These studies have shown that while DNA polymerases delta, epsilon and alpha are discrete protiens, they must share common structural features by virtue of the ability of several of our monoclonal antibodies to exhibit cross-reactivity. A second approach we have taken is the molecular cloning of human DNA polymerase delta and epsilon. We have cloned the DNA polymerase delta cDNA, and this has allowed us to compare its primary structure to those of human polymerase alpha and other members of this polymerase family. Multiple sequence alignments have revealed that human DNA polymerase delta is also closely related to the herpes virus family of DNA polymerases. In situ hybridization has shown that the human DNA polymerase delta gene is localized to chromosome 19 q13.3–q13.4. In order to further determine the functional regions of the DNA polymerase δ structure we are currently expressing human pol δ inE. coli and baculovirus systems. Other work in our laboratory is directed toward examining the expression of DNA polymerase δ during the cell cycle.     

Replication of UV-damaged DNA: new insights into links between DNA polymerases, mutagenesis and human disease

The existence of homologous genes in diverse species is intriguing. A detailed comparison of the structure and function of gene families may provide important insights into gene regulation and evolution. An unproven assumption is that homologous genes have a common ancestor. During evolution, the original function of the ancestral gene might be retained in the different species which evolved along separate courses. In addition, new functions could have developed as the sequence began to diverge. This may also explain partly the presence of multipurpose genes, which have multiple functions at different stages of development and in different tissues. The Drosophila gene snail is a multipurpose gene; it has been demonstrated that snail is critical for mesoderm formation, for CNS development, and for wing cell fate determination. The related vertebrate Snail and Slug genes have also been proposed to participate in mesoderm formation, neural crest cell migration, carcinogenesis, and apoptosis. In this review, we will discuss the Snail/Slug family of regulators in species ranging from insect to human. We will present the protein structures, expression patterns, and functions based on molecular genetic analyses. We will also include the studies that helped to elucidate the molecular mechanisms of repression and the relationship between the conserved and divergent functions of these genes. Moreover, the studies may enable us to trace the evolution of this gene family.     

Mutational specificities of brominated DNA adducts catalyzed by human DNA polymerases

Chronic inflammation is known to lead to an increased risk for the development of cancer. Under inflammatory condition, cellular DNA is damaged by hypobromous acid, which is generated by myeloperoxidase and eosinophil peroxidase. The reactive brominating species induced brominated DNA adducts such as 8-bromo-2′-deoxyguanosine (8-Br-dG), 8-bromo-2′-deoxyadenosine (8-Br-dA), and 5-bromo-2′-deoxycytidine (5-Br-dC). These DNA lesions may be implicated in carcinogenesis. In this study, we analyzed the miscoding properties of the brominated DNA adducts generated by human DNA polymerases (pols). Site-specifically modified oligodeoxynucleotides containing a single 8-Br-dG, 8-Br-dA, or 5-Br-dC were used as a template in primer extension reactions catalyzed by human pols α, κ, and η. When 8-Br-dG-modified template was used, pol α primarily incorporated dCMP, the correct base, opposite the lesion, along with a small amount of one-base deletion (4.8%). Pol κ also promoted one-base deletion (14.2%), accompanied by misincorporation of dGMP (9.5%), dAMP (8.0%), and dTMP (6.1%) opposite the lesion. Pol η, on the other hand, readily bypassed the 8-Br-dG lesion in an error-free manner. As for 8-Br-dA and 5-Br-dC, all the pols bypassed the lesions and no miscoding events were observed. These results indicate that only 8-Br-dG, and not 5-Br-dC and 8-Br-dA, is a mutagenic lesion; the miscoding frequency and specificity vary depending on the DNA pol used. Thus, hypobromous acid-induced 8-Br-dG adduct may increase mutagenic potential at the site of inflammation.     

DNA polymerases and carcinogenesis

There are many various chromosomal and gene mutations in human cancer cells. The total mutation rate in normal human cells is 2·10⁻⁷ mutations/gene/division. From 6 to 12 carcinogenic mutations can arise by the end of the life, and these can affect the structure of ∼150 protooncogenes and genes encoding suppressors of tumor growth. However, this does not explain the tens and hundreds of thousands of mutations detectable in cancer cells. Mutation is any change of nucleotide sequence in cellular DNA. Gene mutations are mainly consequences of errors of DNA polymerases, especially of their specialized fraction (inaccurate DNA polymerases β, ζ, η, θ, ι, κ, λ, μ, σ, ν, Rev1, and terminal deoxynucleotidyl transferase, and only polymerases θ and σ manifest a slight 3′-exonuclease activity) and also consequences of a decrease in the rate of repair of these errors. Inaccurate specialized human polymerases are able to synthesize DNA opposite lesions in the DNA template, but their accuracy is especially low during synthesis on undamaged DNA. In the present review fundamental features of such polymerases are considered. DNA synthesis stops in the area of its lesion, but this block is overcome due to activities of inaccurate specialized DNA polymerases. After the lesion is bypassed, DNA synthesis is switched to accurate polymerases α, δ, ɛ, or γ. Mechanisms of direct and reverse switches of DNA polymerases as well as their modifications during carcinogenesis are discussed.     

DNA polymerases and repair synthesis in NER in human cells

The late steps of nucleotide excision repair, following incisions to remove the damaged section of DNA, comprise repair synthesis and ligation. In vitro and in vivo studies have shown the size of the repaired patch to be about 30 nucleotides. In vitro studies implicated the replicative polymerases in repair synthesis, but recent in vivo data have shown that several DNA polymerases and ligases are involved in these steps in human cells.     

Effect of aphidicolin on viral and human DNA polymerases.

DNA polymerases induced by Herpes simplex and Vaccinia viruses are inhibited by aphidicolin and this inhibition is probably the basis of its antiviral activity $\text{in vivo}$. Its possible clinical use is however hampered by the concomitant effect on human replicative DNA polymerase α. The inhibition of human α-polymerase is reversible both $\text{in}$$\text{vitro}$ and $\text{in vivo}$ and the changes in the rate of incorporation of thymidine into DNA, following treatment with aphidicolin for a generation time, indicate the likely synchronization of the cells due to this agent. DNA polymerase β, which has recently been shown to carry out repair synthesis of damaged nuclear DNA, is not inhibited by aphidicolin either $\text{in vitro}$ on $\text{in vivo}$ suggesting that the drug could allow a rapid and simple evaluation of DNA repair synthesis due to DNA polymerase β.     

Translesion synthesis past estrogen-derived DNA adducts by human DNA polymerases eta and kappa

Newly discovered human DNA polymerase (pol) eta and kappa are highly expressed in the reproductive organs, such as testis, ovary, and uterus, where steroid hormones are produced. Because treatment with estrogen increases the risk of developing breast, ovary, and endometrial cancers, miscoding events occurring at model estrogen-derived DNA adducts were explored using pol eta and a truncated form of human pol kappa (pol kappaDeltaC). These enzymes bypassed N(2)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyguanosine (dG-N(2)-3MeE) and N(6)-[3-methoxyestra-1,3,5(10)-trien-6-yl]-2'-deoxyadenosine (dA-N(6)-3MeE), which were embedded in site-specifically modified oligodeoxynucleotide templates. Quantitative analysis of base substitutions and deletions occurring at the lesion site showed that pol kappaDeltaC was more efficient at incorporating dCMP opposite the dG-N(2)-3MeE lesion than pol eta. Surprisingly, the frequency of translesion synthesis beyond the dC*dG-N(2)-3MeE pair was 13% of the normal dC*dG pair and was 4 and 6 orders of magnitude higher than that of dC*(+)-trans-dG-N(2)-benzo[a]pyrene and dC*dG-C8-acetylaminofluorene pairs, respectively, suggesting that dG-N(2)-3MeE is a natural substrate for pol kappa. In contrast, the bypass frequency beyond the dT*dA-N(6)-3MeE pair was 7 orders of magnitude less than that for the normal dT*dA pair. dA-N(6)-3MeE is a more miscoding lesion than dG-N(2)-3MeE. Pol eta promoted incorporation of dAMP and dCMP at the dA-N(6)-3MeE lesion, while with pol kappaDeltaC, deletions were more frequently observed, along with incorporation of dAMP and dCMP opposite the lesion. These observations were also supported by steady-state kinetic studies. When taken together, the properties of pol eta and kappa are consistent with the mutagenic events attributed to estrogen-derived DNA adducts.     

Eukaryotic DNA polymerases

The biological properties, classification and phylogeny of eukaryotic DNA polymerases are reviewed.     

Eukaryotic DNA polymerases

Knowledge about eukaryotic DNA polymerases has increased considerably during recent years. Much have been learnt about both the structures and the functions of "classical" DNA polymerases alpha, beta, delta, epsilon and gamma. New DNA polymerases that possess very unusual functions have been identified. They are able to perform translesional synthesis, take part in somatic hypermutation and prevent some cancers. Much attention has also been devoted to the role of 3'-->5' exonuclease activity in the accuracy of DNA synthesis. On the other hand, it have been shown that there are also negative aspects of the activity of DNA polymerases. Lack of some DNA polymerases or even their altered functions may lead to carcinogenesis and accelerate the process of ageing.     

Translesion synthesis past tamoxifen-derived DNA adducts by human DNA polymerases eta and kappa

The long-term treatment of tamoxifen (TAM), widely used for adjuvant chemotherapy and chemoprevention for breast cancer, increases a risk of developing endometrial cancer. A high frequency of K-ras mutations has been observed in the endometrium of women treated with TAM. Human DNA polymerase (pol) eta and pol kappa are highly expressed in the reproductive organs and are associated with translesion synthesis past bulky DNA adducts. To explore the miscoding properties of alpha-(N2-deoxyguanosinyl)tamoxifen (dG-N2-TAM), a major TAM-DNA adduct, site-specifically modified oligodeoxynucleotides containing a single diastereoisomer of trans or cis forms of dG-N2-TAM were prepared by phosphoramidite chemical procedure and used as templates. The primer extension reaction catalyzed by pol kappa deltaC, a truncated form of pol kappa, extended more efficiently past the adduct than that of pol eta by incorporating dCMP, a correct base, opposite the adduct. With pol eta, all diastereoisomers of dG-N2-TAM promoted small amounts of direct incorporation of dAMP and deletions. With pol kappa deltaC, dG-N2-TAM promoted small amounts of dTMP and/or dAMP incorporations and deletions. The miscoding properties varied depending on the diastereoisomer of dG-N2-TAM adducts and the DNA pol used. Steady-state kinetic studies were also performed using either the nonspecific sequence or the K-ras gene sequence containing a single dG-N2-TAM at the second base of codon 12. With pol eta, the bypass frequency past the dA x dG-N2-TAM pair positioned in the K-ras sequence was only 2.3 times lower than that for the dC x dG-N2-TAM pair, indicating that dG-N2-TAM in the K-ras sequence has higher miscoding potential than that in the nonspecific sequence. However, with pol kappa deltaC, the bypass frequency past the dC x dG-N2-TAM pair was higher than that of the dT x dG-N2-TAM pair in both sequences. The properties of pol eta and pol kappa are consistent with the mutagenic events attributed to TAM-DNA adducts.     

DNA polymerases from non stimulated and phytohemagglutinin stimulated normal human lymphocytes

The presence and some properties of DNA polymerases isolated from normal human lymphocytes, non stimulated and stimulated by phytohemagglutinin, are described. In the non stimulated lymphocytes two cytoplasmic DNA polymerases are found, one eluting from DEAE cellulose at 0.07 M NaCl (CI_n) and the other at 0.13 M NaCl (CII_n). In the nuclear soluble fraction only one enzyme activity is found (NI_n) which does not adsorb to DEAE cellulose. In the cytoplasm of stimulated lymphocytes only one enzyme activity is detected (CI_s) which elutes from DEAE cellulose at 0.12 M NaCl. The nuclear soluble fraction contains two activities, NI_s, which does not adsorb to DEAE cellulose, and NII_s, which elutes from DEAE cellulose at 0.07 M NaCl. Some properties of the different enzymes are described which indicate that NI_n and NI_s enzymes are clearly different from the others.     

Replicative DNA polymerases

SUMMARY: Replicative DNA polymerases are essential for the replication of the genomes of all living organisms. On the basis of sequence similarities they can be classified into three types. Type A polymerases are homologous to bacterial polymerases I, Type B comprises archaebacterial DNA polymerases and eukaryotic DNA polymerase alpha, and the bacterial polymerase III class make up type C. Structures have been solved for several type A and B polymerases, which share a similar architecture. The structure of type C is not yet known. The catalytic mechanism of all three types involves two metal-ion-binding acidic residues in the active site. Replicative polymerases are constitutively expressed, but their activity is regulated through the cell cycle and in response to different growth conditions.     

Two novel human and mouse DNA polymerases of the polX family

We describe here two novel mouse and human DNA polymerases: one (pol λ) has homology with DNA polymerase β while the other one (pol µ) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix–loop–helix DNA-binding motifs and polymerase X domain. mRNA expression of pol λ is highest in testis and fetal liver, while expression of pol µ is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol µ gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, γ-rays or H₂O₂). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.