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PubMed1992

Decreased fidelity of DNA polymerases and decreased DNA excision repair in aging mice: effects of caloric restriction.

Hepatic DNA polymerases from calorie restricted and ad libitum 26 month old C57BL/6 mice showed a decline in fidelity of nucleotide incorporation compared with weanling animals. Both alpha and beta polymerases from calorie restricted aged mice exhibited a higher level of fidelity than polymerases from ad libitum aged mice. UV-initiated unscheduled DNA synthesis was significantly higher in hepatocytes from weanling and 18 month old calorie restricted animals compared with cells from 18 month old ad libitum animals, while MMS-initiated unscheduled DNA synthesis did not differ significantly between cells from young and old or ad libitum and calorie restricted animals. These data suggest that calorie restriction could play a significant role in decreasing the age-related decline of cellular mechanisms expected to reduce the rate at which mutations accumulate during aging, and could potentially prolong the onset age of mutation-associated diseases of the elderly.

Aging↗
PubMed1987

A new approach to chemotherapy based on molecular biology and nucleic acid chemistry: Matagen (masking tape for gene expression).

The nucleotide sequences of genes contain information which can potentially be used to understand gene function and thus the biological properties of living organisms. This information can also be used to develop innovative new strategies for chemotherapy employing sequence-specific non-ionic oligonucleoside methylphosphonates. These oligonucleotide analogs, termed Matagen (an acronym for masking tape for gene expression), have the following properties: (1) the negatively charged phosphodiester linkage normally found in nucleic acids is replaced with a non-charged methylphosphonate group which confers increased lipophilicity to the oligomer; (2) the oligomers form stable hydrogen-bonded complexes with complementary nucleic acid sequences and retain the fidelity of Watson-Crick base pairing; (3) the lipophilic oligomers cross the cell membrane and also enter various organs of the body; and (4) the methylphosphonate backbone is inherently resistant to nuclease hydrolysis and thus oligomers are taken up intact from cell culture media and remain stable within the cellular environment. Two general strategies are used to block gene expression by Matagens at the mRNA level in mammalian cells. In the first approach, Matagens complementary to specific sites such as the initiation codon region are used to block translation of mRNA. Thus Matagens specifically inhibit translation of rabbit globin mRNA in cell-free systems and rabbit reticulocytes, and vesicular stomatitis virus protein synthesis, but not cellular protein synthesis, in virus-infected cells. In the second approach, Matagens complementary to splice junctions of precursor mRNAs are used to inhibit splicing. For example, a Matagen complementary to the donor splice junction of simian virus 40 (SV40) large T-antigen mRNA inhibits T-antigen synthesis in SV40-infected cells, and a Matagen complementary to the acceptor splice junction of herpes simplex virus (HSV) immediate early pre-mRNA 4 + 5 inhibits HSV replication in virus-infected cells. Two new types of Matagen, one derivatized with the photoactivatable cross-linking group psoralen and the other derivatized with a hydroxyl radical-producing group, EDTA-Fe(II), have been designed to improve the efficacy of Matagen and to overcome some of the problems inherent in physical binding of Matagens to complementary nucleic acids. The Matagen approach provides a new way to design antiviral and chemotherapeutic agents in a rational manner. It combines nucleic acid chemistry and chemotherapy to form a common basis for drug development as well as to provide fundamental knowledge about organisms and humans.

Antineoplastic Agents↗
PubMed1986

Evidence for an intermediate in DNA synthesis involving pyrophosphate exchange. A possible role in fidelity.

The incorporation of exogenous deoxyribonucleotide monophates (dNMP) was measured under conditions of ongoing DNA synthesis, providing arguments for the existence of a [DNAn X dNMP X PPi] intermediate in the nucleotide incorporation step of DNA synthesis: (formula; see text). The existence of such an intermediate is suggested by an apparent exchange of both dNMP and pyrophosphate (PPi) moieties of the deoxyribonucleotide triphosphate (dNTP) substrate with exogenous molecules. Such exchange and the incorporation of exogenous dNMP into DNA, strictly require ongoing DNA synthesis, suggesting that the energy for exchange reactions is provided by the cleavage of dNTP substrate. We propose that nucleotide selection during ongoing DNA synthesis results largely from the different relative rates of forward (beta) and backward (-alpha) reactions involving the [DNAn X dNMP X PPi] intermediate: the forward (incorporation) reaction is expected to predominate for the correct nucleotide, whereas the backward (abortive) reaction is expected to predominate for incorrect nucleotides.

Animals↗
PubMed1993

Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization.

Phi 29 DNA polymerase is able to catalyze two different synthetic reactions: protein-primed initiation and DNA polymerization. We have studied the fidelity of phi 29 DNA polymerase when carrying out these two reactions. Global fidelity was dissected into three steps: insertion discrimination, mismatch elongation, and proofreading. The insertion discrimination of phi 29 DNA polymerase in DNA polymerization ranged from 10(4) to 10(6). The efficiency of mismatch elongation was 10(5)-10(-6)-fold lower than that of a properly paired primer terminus. These factors indicate that DNA polymerization catalyzed by phi 29 DNA polymerase is a highly accurate process. Conversely, the insertion fidelity of protein-primed initiation was quite low, the insertion discrimination factor being about 10(2). Mismatch elongation discrimination was also rather low: mismatched terminal protein (TP).dNMP complexes were elongated from 2- to 6-fold more slowly than the correct TP.dNMP complex. Even more, the 3'-->5' exonuclease activity of phi 29 DNA polymerase was unable to act on the TP.dNMP initiation complex, precluding the possibility that a wrong dNMP covalently linked to TP could be excised and corrected. Therefore, protein-primed initiation can be predicted as a quite inaccurate reaction. The problem of maintaining the sequence at the DNA ends is discussed in the context of a recently described model for protein-primed initiation.

Bacillus Phages↗
PubMed2002

DNA repair in terminally differentiated cells.

Terminally differentiated cells do not replicate their genomic DNA, and could therefore dispense with the task of removing DNA damage from the non-essential bulk of their genome, as long as they are able to maintain the integrity of the genes that must be expressed. There is increasing experimental evidence that this is indeed the case, at least for some repair pathways such as nucleotide excision repair (NER). In this review, we examine a number of terminally differentiated cell systems in which it has been demonstrated that DNA repair is attenuated at the global genome level, but maintained in expressed genes. How these cells manage to repair transcribed genes is not yet fully elucidated, but there are indications that the transcription-coupled repair (TCR) pathway could maintain integrity of the transcribed strand (TS) in the active genes. We have observed in neurons that the non-transcribed strand (NTS) of active genes is also well repaired, a phenomenon that we have named differentiation-associated repair (DAR). It is conceivable that DAR is necessary to maintain the integrity of the template strand that is needed by TCR to complete the repair of lesions in the TS of essential expressed genes with high fidelity.

Animals↗
PubMed1997

Mispair extension fidelity of human immunodeficiency virus type 1 reverse transcriptases with amino acid substitutions affecting Tyr115.

The role of Tyr115 of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) in the mispair extension fidelity of DNA dependent DNA synthesis was analysed by using a series of 15 mutant enzymes with substitutions at Tyr115. Their kinetic parameters for elongation using homopolymeric RNA-DNA and heteropolymeric DNA-DNA complexes showed major effects of the amino acid substitutions on the Km value for dNTP. Enzymes with large hydrophobic residues at position 115 displayed lower Km values than enzymes with small and charged amino acids at this position. The influence of all these amino acid replacements in mispair extension fidelity assays was analyzed using three different mismatches (A:C, A:G and A:A) at the 3'-terminal position of the primer DNA. For the A:C mispair, a 2. 6-33.4-fold increase in mispair extension efficiency (fext) was observed as compared with the wild-type enzyme. Unexpectedly, all the mutants tested as well as the wild-type RT were very efficient in extending the A:G and A:A transversion mispairs. This effect was due to the template-primer sequence context and not to the buffer conditions of the assay. The data support a role of Tyr115 in accommodating the complementary nucleotide into the nascent DNA while polymerization takes place.

DNA Repair↗
PubMed1996

Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo[a]pyrenediol epoxide-modified DNA.

DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur. Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome. The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest. The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase. We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine. However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

Bacteriophage T7↗
PubMed2000

In vivo retroviral integration: fidelity to size of the host DNA duplication might Be reduced when integration occurs near sequences homologous to LTR ends.

Integrated retroviral DNAs are flanked by a short duplication of target DNA whose size is virus specific and invariable. We have sequenced the junctions between an ALSV (Avian Leukemia and Sarcoma Viruses)-based vector and quail DNA from five individual proviruses. Three proviruses were flanked by the expected 6-bp duplication of host DNA, whereas the two others were flanked by a 5-bp duplication. Nucleotide sequencing of the native integration sites of these two proviruses showed that these integrations had occurred at the immediate vicinity of either a CA or a TG dinucleotide, revealing striking microhomologies between the integration sites and viral LTR ends. These results suggest that size duplication of the target DNA might be influenced by nucleotidic sequence at the site of integration.

Animals↗
PubMed1988

Viral proteases: an emerging therapeutic target.

Only a few viral diseases are presently treatable because of our limited knowledge of specific viral target molecules. An attractive class of viral molecules toward which chemotherapeutic agents could be aimed are proteases coded by some virus groups such as retro- or picornaviruses (poliomyelitis, common cold virus). The picornavirus enzymes were discovered first, and they have now been characterized by a combination of molecular-genetic and biochemical approaches. Several laboratories have expressed the picornaviral enzymes in heterologous systems and have reported proteolytic activity, as well as the high cleavage fidelity diagnostic of the viral proteases. After dealing with several technical difficulties often encountered in standard genetic engineering approaches, one viral protease is now available to us in quantity and is amendable to mutagenic procedures. The initial outcome of the mutagenesis studies has been the confirmation of our earlier work with inhibitors, which suggested a cysteine active-site class. There is a clustering of active-site residues which may be unique to these viruses. The requirement for an active-site cysteine-histidine pair in combination with detailed information on the viral cleavage sites has permitted design of selective inhibitors with attractive antiviral properties. Future goals include investigation of the structural basis for selective processing and application of the cleavage specificity to general problems in genetic engineering.

Humans↗
PubMed1988

Specificity of proofreading by the 3'----5' exonuclease of the DNA polymerase-primase of Drosophila melanogaster.

The DNA polymerase-primase from Drosophila melanogaster contains a cryptic 3'----5' exonuclease that can be detected after separation of the 182-kDa polymerase subunit from the four-subunit enzyme. To determine the specificity of excision of mispaired nucleotides by the exonuclease, we have utilized primed phi X174am3 single-stranded DNA containing a noncomplementary nucleotide at the 3'-primer terminus, opposite deoxyadenosine at position 587 in the amber3 codon of the template strand. In the absence of polymerization, the preference for excision of the mispaired nucleotide from the primer is C greater than A much greater than G. Excision under these conditions is inhibited by the addition of deoxyguanosine monophosphate. Under conditions of concomitant DNA synthesis, the preference for excision at this site becomes A = G much greater than C, and excision is insensitive to deoxyguanosine monophosphate. The high fidelity of DNA synthesis exhibited by the isolated 182-kDa polymerase subunit is not reduced by concentrations of deoxyguanosine monophosphate or adenosine monophosphate that inhibit proofreading by prokaryotic DNA polymerases. Thus, the 3'----5' exonuclease of the Drosophila DNA polymerase-primase participates in exonucleolytic proofreading by excising noncomplementary nucleotides prior to extension of the primer by polymerase action. The deoxynucleoside triphosphate analogs N2-(p-butylphenyl)deoxyguanosine triphosphate and N2-(p-butylphenyl)deoxyadenosine triphosphate are potent inhibitors of DNA polymerase alpha. Like calf thymus DNA polymerase delta, recently determined to have proofreading capability, DNA synthesis by the isolated Drosophila 182-kDa polymerase subunit was not inhibited by the two analogs. In contrast, DNA synthesis by the intact Drosophila polymerase-primase complex was inhibited greater than 95% by these analogs.

Animals↗
PubMed2006

Exogenous 8-oxo-dG is not utilized for nucleotide synthesis but enhances the accumulation of 8-oxo-Gua in DNA through error-prone DNA synthesis.

7,8-Dihydro-8-oxoguanine (8-oxo-Gua) and its nucleoside in cytosol are derived from the repair of oxidative DNA and the cleanup of oxidatively damaged DNA precursors, respectively. While the harmful effects of 8-oxo-Gua present in DNA have been studied extensively, few have reported its effects on cytosolic function. Our previous study showed that the addition of 8-oxo-dG to culture media caused an accumulation of 8-oxo-Gua in nuclear DNA in several leukemic cells including KG-1, which lack 8-oxoguanine glycosylase 1 (OGG1) activity due to mutational loss. However, the mechanism underlying 8-oxo-Gua level increases in DNA has not been addressed. In this study, we elucidated the metabolic fate of 8-oxo-Gua-containing nucleotide and the effect of exogenous 8-oxo-dG on DNA synthesis in KG-1 cells. We found that 8-oxo-dGMP was rapidly dephosphorylated to 8-oxo-dG rather than phosphorylated to 8-oxo-dGDP, thus indicating that 8-oxo-Gua-containing molecule is not used as a substrate for DNA synthesis in KG-1 cells. In fact, radiolabeled 8-oxo-dG was incubated but radioactivity was not detected in nuclear DNA of KG-1 cells, showing that 8-oxo-dG is not directly incorporated into DNA. Interestingly, the activity of DNA polymerase beta, which synthesize DNA with low fidelity increased in KG-1 cells treated with 8-oxo-dG, whereas the expression of DNA polymerase alpha decreased. In addition, the accumulation of 8-oxo-Gua in KG-1 DNA was completely inhibited by a specific inhibitor of DNA polymerase beta. Thus, our findings address that the insertion of 8-oxo-dG into KG-1 DNA is not due to the direct incorporation of exogenous 8-oxo-dG, but rather to the inaccurate incorporation of endogenous 8-oxo-dGTP by DNA polymerase beta. It further suggests that 8-oxo-dG in the cytosol may function as an active molecule itself and perturb the well-defined DNA synthesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Quantum-chemical ab initio study on the adenine-difluorotoluene complex--a mimic for the adenine-thymine base pair.

Recent experiments have shown that difluorotoluene (F), a nonpolar isostere for thymine (T), codes efficiently and specifically for adenine (A) in DNA replication. F has almost the same shape as thymine but it is unable to form conventional hydrogen bonds with adenine. Therefore, it has been claimed that not hydrogen bonding but shape complementary may be important for the selection of the correct bases by DNA-replicating enzymes. In order to gain deeper insight into structure, charge distribution and energetics of the A-F and A-T base pairs we have performed quantum-chemical ab initio and density functional calculations at the HF, MP2 and B3LYP levels. The interaction energy of the A-F complex amounts to -3.8 kcal/mol (MP2) and is thus substantially smaller than typical ab initio interaction energies for Watson-Crick or non-canonical base pairs. The A-T and A-F complexes are planar and their overall geometries are similar (root-mean-square deviation: 0.4 A). The calculated donor acceptor atom distances in A-T are in good agreement with the experimental mean values obtained from an analysis of 21 high resolution DNA structures. However, A-F shows a base pair opening as compared to A-T. Even though the interaction energy in the A-F base pair is small, the distances for the N6-H...F and N1...H-C3 contacts are still below the sum of the van-der-Waals radii, which means that the interaction is not governed by van-der-Waals forces alone. If the experimental findings can be confirmed, then our results indicate that DNA polymerase is able to retain high fidelity with base pairs of much smaller interaction energies than found for the conventional Watson-Crick and non-canonical base pairs.

Adenine↗
PubMed2002

Active site mutation in DNA polymerase gamma associated with progressive external ophthalmoplegia causes error-prone DNA synthesis.

Progressive external ophthalmoplegia (PEO) is a heritable mitochondrial disorder characterized by the accumulation of multiple point mutations and large deletions in mtDNA. Autosomal dominant PEO was recently shown to co-segregate with a heterozygous Y955C mutation in the human gene encoding the sole mitochondrial DNA polymerase, DNA polymerase gamma (pol gamma). Since Tyr-955 is a highly conserved residue critical for nucleotide recognition among family A DNA polymerases, we analyzed the effects of the Y955C mutation on the kinetics and fidelity of DNA synthesis by the purified human mutant polymerase in complex with its accessory subunit. The Y955C enzyme retains a wild-type catalytic rate (k(cat)) but suffers a 45-fold decrease in apparent binding affinity for the incoming nucleoside triphosphate (K(m)). The Y955C derivative is 2-fold less accurate for base pair substitutions than wild-type pol gamma despite the action of intrinsic exonucleolytic proofreading. The full mutator effect of the Y955C substitution was revealed by genetic inactivation of the exonuclease, and error rates for certain mismatches were elevated by 10-100-fold. The error-prone DNA synthesis observed for the Y955C pol gamma is consistent with the accumulation of mtDNA mutations in patients with PEO.

Amino Acid Sequence↗
PubMed1995

Three-dimensional quantitative autoradiography by disparity analysis: theory and application to image averaging of local cerebral glucose utilization.

Traditional autoradiographic image analysis has been restricted to the two-dimensional assessment of local cerebral glucose utilization (LCMRglc) or blood flow in individual brains. It is advantageous, however, to generate an entire three-dimensional (3D) data set and to develop the ability to map replicate images derived from multiple studies into the same 3D space, so as to generate average and standard deviation images for the entire series. We have developed a novel method, termed "disparity analysis," for the alignment and mapping of autoradiographic images. We present the theory of this method, which is based upon a linear affine model, to analyze point-to-point disparities in two images. The method is a direct one that estimates scaling, translation, and rotation parameters simultaneously. Disparity analysis is general and flexible and deals well with damaged or asymmetric sections. We applied this method to study LCMRglc in nine awake male Wistar rats by the [14C]2-deoxyglucose method. Brains were physically aligned in the anteroposterior axis and were sectioned subserially at 100-microns intervals. For each brain, coronal sections were aligned by disparity analysis. The nine brains were then registered in the z-axis with respect to a common coronal reference level (bregma + 0.7 mm). Eight of the nine brains were mapped into the remaining brain, which was designated the "template," and aggregate 3D data sets were generated of the mean and standard deviation for the entire series. The averaged images retained the major anatomic features apparent in individual brains but with some defocusing. Internal anatomic features of the averaged brain were smooth, continuous, and readily identifiable on sections through the 3D stack. The fidelity of the internal architecture of the averaged brain was compared with that of individual brains by analysis of line scans at four representative levels. Line scan comparisons between corresponding sections and their template showed a high degree of correlation, as did similar comparisons performed on entire sections. Fourier analysis of line scan data showed retention of low-frequency information with the expected attenuation of high-frequency components produced by averaging. Region-of-interest (ROI) analysis of the averaged brain yielded LCMRglc values virtually identical to those derived from measurements and subsequent averaging of data from individual brains.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

Cleavage of substrates with mismatched nucleotides by Flap endonuclease-1. Implications for mammalian Okazaki fragment processing.

Flap endonuclease-1 (FEN1) is proposed to participate in removal of the initiator RNA of mammalian Okazaki fragments by two pathways. In one pathway, RNase HI removes most of the RNA, leaving a single ribonucleotide adjacent to the DNA. FEN1 removes this ribonucleotide exonucleolytically. In the other pathway, FEN1 removes the entire primer endonucleolytically after displacement of the 5'-end region of the Okazaki fragment. Cleavage would occur beyond the RNA, a short distance into the DNA. The initiator RNA and an adjacent short region of DNA are synthesized by DNA polymerase alpha/primase. Because the fidelity of DNA polymerase alpha is lower than that of the DNA polymerases that complete DNA extension, mismatches occur relatively frequently near the 5'-ends of Okazaki fragments. We have examined the ability of FEN1 to repair such errors. Results show that mismatched bases up to 15 nucleotides from the 5'-end of an annealed DNA strand change the pattern of FEN1 cleavage. Instead of removing terminal nucleotides sequentially, FEN1 appears to cleave a portion of the mismatched strand endonucleolytically. We propose that a mismatch destabilizes the helical structure over a nearby area. This allows FEN1 to cleave more efficiently, facilitating removal of the mismatch. If mismatches were not introduced during synthesis of the Okazaki fragment, helical disruption would not occur, nor would unnecessary degradation of the 5'-end of the fragment.

Animals↗
PubMed2006

Human DNA polymerase N (POLN) is a low fidelity enzyme capable of error-free bypass of 5S-thymine glycol.

Human DNA polymerase N (POLN or pol nu) is the most recently discovered nuclear DNA polymerase in the human genome. It is an A-family DNA polymerase related to Escherichia coli pol I, human POLQ, and Drosophila Mus308. We report the first purification of the recombinant enzyme and examination of its biochemical properties, as a step toward understanding the functions of POLN. Unusual for an A-family DNA polymerase, POLN is a low fidelity enzyme incorporating T opposite template G with a frequency of 0.45 and G opposite template T with a frequency of 0.021. The frequency of misincorporation of T opposite template G is higher than any other known DNA polymerase. POLN has a processivity of DNA synthesis (1-100 nucleotides) similar to the exonuclease-deficient Klenow fragment of E. coli pol I, is inhibited by dideoxynucleotides, and resistant to aphidicolin. The strand displacement activity of POLN was higher than exonuclease-deficient Klenow fragment. Furthermore, POLN can perform translesion synthesis past thymine glycol, a common endogenous and radiation-induced product of reactive oxygen species damage to DNA. Thymine glycol blocks DNA synthesis by most DNA polymerases, but POLN was particularly adept at efficient and accurate translesion synthesis past a 5S-thymine glycol.

Amino Acid Sequence↗
PubMed2006

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins.

DNA polymerase zeta (pol zeta) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol zeta alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol zeta. Pol zeta is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol zeta is substantially lower than that of homologous B family pols alpha, delta and epsilon. Pol zeta is particularly error prone for substitutions in specific sequence contexts and generates multiple single base errors clustered in short patches at a rate that is unprecedented in comparison with other polymerases. The unique error specificity of pol zeta in vitro is consistent with Pol zeta-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex mutations observed here, indicates that pol zeta contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them.

Base Sequence↗
PubMed1997

The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity.

BACKGROUND: Cell cycle checkpoints maintain the fidelity of the somatic cell cycle by ensuring that one step in the cell cycle is not initiated until a previous step has been completed. The extent to which cell cycle checkpoints play a role in the initial rapid embryonic divisions of higher eukaryotes is unclear. The initial syncytial divisions of Drosophila embryogenesis provide an excellent opportunity to address this issue as they are amenable to both genetic and cellular analysis. In order to study the relevance of cell cycle checkpoints in early Drosophila embryogenesis, we have characterized the maternal-effect grapes (grp) mutation, which may affect feedback control during early syncytial divisions. RESULTS: The Drosophila grp gene encodes a predicted serine/threonine kinase and has significant homology to chk1/rad27, a gene required for a DNA damage checkpoint in Schizosaccharomyces pombe. Relative to normal embryos, embryos derived from grp-mutant mothers exhibit elevated levels of DNA damage. During nuclear cycles 12 and 13, alignment of the chromosomes on the metaphase plate was disrupted in grp-derived embryos, and the embryos underwent a progression of cytological events that were indistinguishable from those observed in normal syncytial embryos exposed to X-irradiation. The mutant embryos also failed to progress through a regulatory transition in Cdc2 activity that normally occurs during interphase of nuclear cycle 14. CONCLUSION: We propose that the primary defect in grp-derived embryos is a failure to replicate or repair DNA completely before mitotic entry during the late syncytial divisions. This suggests that wild-type grp functions in a developmentally regulated DNA replication/damage checkpoint operating during the late syncytial divisions. These results are discussed with respect to the proposed function of the chk1/rad27 gene.

Amino Acid Sequence↗