Molecular consequences of the loss of 3'→5' exonuclease activity of DNA polymerases delta and epsilon

The DNA replication polymerases δ and ϵ have an inherent proofreading mechanism in the form of a 3'→5' exonuclease. Upon recognition of errant deoxynucleotide incorporation into DNA, the nascent primer terminus is partitioned to the exonuclease active site where the incorrectly paired nucleotide is excised before resumption of polymerization. The goal of this project was to identify the cellular and molecular consequences of an exonuclease deficiency. The proofreading capability of model system MEFs with EXOII mutations was abolished without altering polymerase function.^ It was hypothesized that 3'→5' exonucleases of polymerases δ and ϵ are critical for prevention of replication stress and important for sensitization to nucleoside analogs. To test this hypothesis, two aims were formulated: Determine the effect of the exonuclease active site mutation on replication related molecular signaling and identify the molecular consequences of an exonuclease deficiency when replication is challenged with nucleoside analogs.^ Via cell cycle studies it was determined that larger populations of exonuclease deficient cells are in the S-phase. There was an increase in levels of replication proteins, cell population growth and DNA synthesis capacity without alteration in cell cycle progression. These findings led to studies of proteins involved in checkpoint activation and DNA damage sensing. Finally, collective modifications at the level of DNA replication likely affect the strand integrity of DNA at the chromosomal level.^ Gemcitabine, a DNA directed nucleoside analog is a substrate of polymerases δ and ϵ and exploits replication to become incorporated into DNA. Though accumulation of gemcitabine triphosphate was similar in all cell types, incorporation into DNA and rates of DNA synthesis were increased in exonuclease defective cells and were not consistent with clonogenic survival. This led to molecular signaling investigations which demonstrated an increase in S-phase cells and activation of a DNA damage response upon gemcitabine treatment.^ Collectively, these data indicate that the loss of exonuclease results in a replication stress response that is likely required to employ other repair mechanisms to remove unexcised mismatches introduced into DNA during replication. When challenged with nucleoside analogs, this ongoing stress response coupled with repair serves as a resistance mechanism to cell death.^

Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase $\alpha$ are the molecular targets for two metal ions, Zn$\sp{2+}$ and Cd$\sp{2+},$ and an anticancer drug, F-ara-ATP.^ Human DNA ligases were purified to homogeneity and their AMP binding domains were mapped. Although their AMP-binding domains are similar, there could be difference between the two ligases in their DNA binding domains.^ The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP.^ A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex.^ F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3$\sp\prime$-terminus of DNA nick by DNA polymerase $\alpha.$^ All steps of the DNA ligation reaction were inhibited by Zn$\sp{2+}$ and Cd$\sp{2+}$ in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn$\sp{2+}$ and Cd$\sp{2+}$ showed their contradictory effects on the fidelity of the reaction by human DNA polymerase $\alpha.$ Zn$\sp{2+}$ decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd$\sp{2+}$ increased the frequencies of both misinsertion and mispair extension at very low concentration. Our data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported. ^

Studies of the expression of DNA repair related genes

This dissertation examines the biological functions and the regulation of expression of DNA ligase I by studying its expression under different conditions.^ The gene expression of DNA ligase I was induced two- to four-fold in S-phase lymphoblastoid cells but was decreased to 15% of control after administration of a DNA damaging agent, 4-nitroquinoline-1-oxide. When cells were induced into differentiation, the expression level of DNA ligase I was decreased to less than 15% of that of the control cells. When the gene of DNA ligase I was examined for tissue specific expression in adult rats, high levels of DNA ligase I mRNA were observed in testis (8-fold), intermediate levels in ovary and brain (4-fold), and low levels were found in intestine, spleen, and liver (1- to 2-fold).^ In confluent cells of normal skin fibroblasts, UV irradiation induced the gene expression of DNA ligase I at 24 and 48 h. The induction of DNA ligase I gene expression requires active p53 protein. Introducing a vector containing the wild type p53 protein in the cells caused an induction of the DNA ligase I protein 24 h after the treatment.^ Our results indicate that, in addition to the regulation by phosphorylation/dephosphorylation, cellular DNA ligase I activity can be regulated at the gene transcription level, and the p53 tumor suppresser is one of the transcription factors for the DNA ligase I gene. Also, our results suggest that DNA ligase I is involved in DNA repair as well as in DNA replication.^ Also, as an early attempt to clone the human homolog of the yeast CDC9 gene which has been shown to be involved in DNA replication, DNA repair, and DNA recombination, we have identified a human gene with mRNA of 1.7 kb. This dissertation studies the gene regulation and the possible biological functions of this new human gene by examining its expression at different stages of the cell cycle, during cell differentiation, and in cellular response to DNA damage.^ The new gene that we recently identified from human cells is highly expressed in brain and reproductive organs (BRE). This BRE gene encodes an mRNA of 1.7-1.9 kb, with an open reading frame of 1,149 bp, and gives rise to a deduced polypeptide of 383 amino acid residues. No extensive homology was found between BRE and sequences from the EMBL-Gene Banks. BRE showed tissue-specific expression in adult rats. The steady state mRNA levels were high in testis (5-6 fold), ovary and brain (3-4 fold) compared to the spleen level, but low in intestine and liver (1-2 fold). The expression of this gene is responsive to DNA damage and/or retinoic acid (RA) treatment. Treatment of fibroblast cells with UV irradiation and 4-nitroquinoline-1-oxide caused more than 90% and 50% decreases in BRE mRNA, respectively. Similar decreases in BRE expression were observed after treatment of the brain glioma cell line U-251 and the promyelocytic cell line HL-60 with retinoic acid. (Abstract shortened by UMI). ^

Investigation of I -compounds (newly discovered indigenous DNA modifications) by different approaches

I-compounds are newly discovered covalent DNA modifications detected by the $\sp{32}$P-postlabeling assay. They are age-dependent, tissue-specific and sex-different. The origin(s), chemistry and function(s) of I-compounds are unknown. The total level of I-compounds in 8-10 month old rat liver is 1 adduct in 10$\sp7$ nucleotides, which is not neglectable. It is proposed that I-compounds may play a role in spontaneous tumorigenesis and aging.^ In the present project, I-compounds were investigated by several different approaches. (1) Dietary modulation of I-compounds. (2) Comparison of I-compounds with persistent carcinogen DNA adducts and 5-methylcytosine. (3) Strain differences of I-compounds in relation to organ site spontaneous tumorigenesis. (4) Effects of nongenotoxic hepatocarcinogenes on I-compounds.^ It was demonstrated that the formation of I-compounds is diet-related. Rats fed natural ingredient diet exhibited more complex I-spot patterns and much higher levels than rats fed purified diet. Variation of major nutrients (carbohydrate, protein and fat) in the diet, produced quantitative differences in I-compounds of rat liver and kidney DNAs. Physiological level of vitamin E in the diet reduced intensity of one I-spot compared with vitamin E deficient diet. However, extremely high level of vitamin E in the diet gave extra spot and enhanced the intensities of some I-spots.^ In regenerating rat liver, I-compounds levels were reduced, as carcinogen DNA adducts, but not 5-methylcytosine, i.e. a normal DNA modification.^ Animals with higher incidences of spontaneous tumor or degenerative diseases tended to have a lower level of I-compounds.^ Choline devoid diet induced a drastic reduction of I-compound level in rat liver compared with choline supplemented diet. I-compound levels were reduced after multi-doses of carbon tetrachloride (CCl$\sb4$) exposure in rats and single dose exposure in mice. An inverse relationship was observed between I-compound level and DNA replication rate. CCl$\sb4$-related DNA adduct was detected in mice liver and intensities of some I-spots were enhanced 24 h after a single dose exposure.^ The mechanisms and explanations of these observations will be discussed. I-compounds are potentially useful indicators in carcinogenesis studies. ^

Analysis of the mechanism of replication inhibition of the tumor suppressor gene p53

p53 plays a role in cell cycle arrest and apoptosis. p53 has also been shown to be involved in DNA replication. To study the effect of p53 on DNA replication, we utilized a SV40 based shuttle vector system. The pZ402 shuttle vector, was constructed with a mutated T-antigen unable to interact with p53 but able to support replication of the shuttle vector. When a transcriptional activation domain p53 mutant was tested for its ability to inhibit DNA replication no inhibition was observed. Competition assays with the DNA binding domain of p53 was also able to block the inhibition of DNA replication by p53 suggesting that p53 can inhibit DNA replication through the transcriptional activation of a target gene. One likely target gene, p21$\sp{\rm cip/waf}$ was tested to determine whether p53 inhibited DNA replication by transcriptionally activating p21$\sp{\rm cip/waf}$. Two independent approaches utilizing p21$\sp{\rm cip/waf}$ null cells or the expression of an anti-sense p21$\sp{\rm cip/waf}$ expression vector were utilized. p53 was able to inhibit pZ402 replication independently of p21$\sp{\rm cip/waf}$. p53 was also able to inhibit DNA replication independent of the p53 target genes Gadd45 and the replication processivity factor PCNA. The inhibition of DNA replication by p53 was also independent of direct DNA binding to a consensus site on the replicating plasmid. p53 mutants can be classified into two categories: conformational and DNA contact mutants. The two types of p53 mutants were tested for their effects on DNA replication. While all conformational mutants were unable to inhibit DNA replication three out of three DNA contact mutants tested were able to inhibit DNA replication. The work here studies the effect wild-type and mutant p53 has on DNA replication and demonstrated a possible mechanism by which wild-type p53 could inhibit DNA replication through the transcriptional activation of a target gene. ^

Crystal structure of a thermostable type B DNA polymerase from Thermococcus gorgonarius

Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe here the 2.5 Å resolution crystal structure of a DNA polymerase from the Archaea Thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function. Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA–protein interface. In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site. A physiological role of this “closed” conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents.

A human Cds1-related kinase that functions downstream of ATM protein in the cellular response to DNA damage

Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. Much of our current understanding of checkpoints comes from genetic studies conducted in yeast. In the fission yeast Schizosaccharomyces pombe (Sp), SpRad3 is an essential component of both the DNA damage and DNA replication checkpoints. The SpChk1 and SpCds1 protein kinases function downstream of SpRad3. SpChk1 is an effector of the DNA damage checkpoint and, in the absence of SpCds1, serves an essential function in the DNA replication checkpoint. SpCds1 functions in the DNA replication checkpoint and in the S phase DNA damage checkpoint. Human homologs of both SpRad3 and SpChk1 but not SpCds1 have been identified. Here we report the identification of a human cDNA encoding a protein (designated HuCds1) that shares sequence, structural, and functional similarity to SpCds1. HuCds1 was modified by phosphorylation and activated in response to ionizing radiation. It was also modified in response to hydroxyurea treatment. Functional ATM protein was required for HuCds1 modification after ionizing radiation but not after hydroxyurea treatment. Like its fission yeast counterpart, human Cds1 phosphorylated Cdc25C to promote the binding of 14-3-3 proteins. These findings suggest that the checkpoint function of HuCds1 is conserved in yeast and mammals.

Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA

Integration of transgenic DNA into the plant genome was investigated in 13 transgenic oat (Avena sativa L.) lines produced using microprojectile bombardment with one or two cotransformed plasmids. In all transformation events, the transgenic DNA integrated into the plant genome consisted of intact transgene copies that were accompanied by multiple, rearranged, and/or truncated transgene fragments. All fragments of transgenic DNA cosegregated, indicating that they were integrated at single gene loci. Analysis of the structure of the transgenic loci indicated that the transgenic DNA was interspersed by the host genomic DNA. The number of insertions of transgenic DNA within the transgene loci varied from 2 to 12 among the 13 lines. Restriction endonucleases that do not cleave the introduced plasmids produced restriction fragments ranging from 3.6 to about 60 kb in length hybridizing to a probe comprising the introduced plasmids. Although the size of the interspersing host DNA within the transgene locus is unknown, the sizes of the transgene-hybridizing restriction fragments indicated that the entire transgene locus must be at least from 35–280 kb. The observation that all transgenic lines analyzed exhibited genomic interspersion of multiple clustered transgenes suggests a predominating integration mechanism. We propose that transgene integration at multiple clustered DNA replication forks could account for the observed interspersion of transgenic DNA with host genomic DNA within transgenic loci.

Translocation and specific cleavage of bacteriophage T7 DNA in vivo by EcoKI

Infection of Escherichia coli containing the type I restriction enzyme EcoKI by bacteriophage T7 0.3 mutants leads to restriction during the late stages of genome entry and during DNA replication. Patterns of cleavage in vivo suggest that some cutting occurs near the midpoint of two recognition sites, consistent with the idea that EcoKI translocates DNA bidirectionally through itself and cuts when two enzyme molecules collide. Rapid ejection of a 0.3+ T7 genome from a bacteriophage λ particle results in degradation of the infecting DNA by EcoKI, showing that the normal T7 DNA translocation process delays restriction. A unique recognition site inserted at the genomic left end allows EcoKI to function as a molecular motor and to translocate the remaining 39 kilobases of T7 DNA into the cell.

Expression of an ortholog of replication protein A1 (RPA1) is induced by gibberellin in deepwater rice

Internodes of deepwater rice are induced to grow rapidly when plants become submerged. This adaptation enables deepwater rice to keep part of its foliage above the rising flood waters during the monsoon season and to avoid drowning. This growth response is, ultimately, elicited by the plant hormone gibberellin (GA). The primary target tissue for GA action is the intercalary meristem of the internode. Using differential display of mRNA, we have isolated a number of genes whose expression in the intercalary meristem is regulated by GA. The product of one of these genes was identified as an ortholog of replication protein A1 (RPA1). RPA is a heterotrimeric protein involved in DNA replication, recombination, and repair and also in regulation of transcription. A chimeric construct, in which the single-stranded DNA-binding domain of rice RPA1 was spliced into the corresponding region of yeast RPA1, was able to complement a yeast rpa1 mutant. The transcript level of rice RPA1 is high in tissues containing dividing cells. RPA1 mRNA levels increase rapidly in the intercalary meristem during submergence and treatment with GA before the increase in the level of histone H3 mRNA, a marker for DNA replication.

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

Funding: Wellcome Trust, 070247/Z/03/A. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

DNA polymerase δ isolated from Schizosaccharomyces pombe contains five subunits

DNA polymerase δ (pol δ) plays an essential role in DNA replication, repair, and recombination. We have purified pol δ from Schizosaccharomyces pombe more than 103-fold and demonstrated that the polymerase activity of purified S. pombe pol δ is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS/PAGE analysis of the purified fraction indicated that the pol δ complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive cdc1-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on S. pombe chromosome 1.

Alternative genetic pathways in colorectal carcinogenesis

The comparative typing of matched tumor and blood DNAs at dinucleotide repeat (microsatellite) loci has revealed in tumor DNA the presence of alleles that are not observed in normal DNA. The occurrence of these additional alleles is possibly due to replication errors (RERs). Although this observation has led to the recognition of a subtype of colorectal cancer with a high incidence of RERs (caused by a deficiency in DNA mismatch repair), a thorough analysis of the RER frequency in a consecutive series of colorectal cancers had not been reported. It is shown here that the extensive typing of 88 colorectal tumors reveals a bimodal distribution for the frequency of RER at microsatellite loci. Within the major mode (75 tumors, RER− subtype), the probability that a locus exhibited instability did not differ significantly among loci and tumors, being 0.02. The subsequent development of a statistical test for an operational discrimination between the RER− and RER+ subtypes indicated that the probability of misclassification did not exceed 0.001 in this series. The frequency of K-ras mutation was found to be equivalent in the two subtypes. However, in the RER+ tumors, the p53 gene mutation was less frequently detected, the adenomatous polyposis coli (APC) mutation was rare, and the biallelic inactivation of either of these genes was not observed. Furthermore, the concomitant occurrence of APC and tumor growth factor β receptor type II gene alterations was found only once. These data suggest that the repertoires of genes that are frequently altered in RER+ and RER− tumors may be more different than previously thought.

The carboxyl terminus of the bacteriophage T4 DNA polymerase is required for holoenzyme complex formation

To further elucidate the mechanism and dynamics of bacteriophage T4 holoenzyme formation, a mutant polymerase in which the last six carboxyl-terminal amino acids are deleted, was constructed, overexpressed, and purified to homogeneity. The mutant polymerase, designated ΔC6 exo−, is identical to wild-type exo− polymerase with respect to kcat, kpol, and dissociation constants for nucleotide and DNA substrate. However, unlike wild-type exo− polymerase, the ΔC6 exo− polymerase is unable to interact with the 45 protein to form the stable holoenzyme. A synthetic polypeptide corresponding to the carboxyl terminus of the wild-type exo− polymerase was tested as an in vitro inhibitor of bacteriophage T4 DNA replication. Surprisingly, the peptide does not directly inhibit holoenzyme complex formation by disrupting the interaction of the polymerase with the 45 protein. On the contrary, the peptide appears to disrupt the interaction of the 44/62 protein with the 45 protein, suggesting that the 44/62 protein and the polymerase use the same site on the 45 protein for functional interactions. Data presented are discussed in terms of a model correlating the functionality of the carboxyl terminus of the polymerase for productive interactions with the 45 protein as well as in terms of the 45 protein concomitantly interacting with the 44/62 protein and polymerase.

A heterodimeric DNA polymerase: Evidence that members of Euryarchaeota possess a distinct DNA polymerase

We describe here a DNA polymerase family highly conserved in Euryarchaeota, a subdomain of Archaea. The DNA polymerase is composed of two proteins, DP1 and DP2. Sequence analysis showed that considerable similarity exists between DP1 and the second subunit of eukaryotic DNA polymerase δ, a protein essential for the propagation of Eukarya, and that DP2 has conserved motifs found in proteins with nucleotide-polymerizing activity. These results, together with our previous biochemical analyses of one of the members, DNA polymerase II (DP1 + DP2) from Pyrococcus furiosus, implicate the DNA polymerases of this family in the DNA replication process of Euryarchaeota. The discovery of this DNA-polymerase family, aside from providing an opportunity to enhance our knowledge of the evolution of DNA polymerases, is a significant step toward the complete understanding of DNA replication across the three domains of life.