Gerald P. Holmquist

Cupric ion/ascorbate/hydrogen peroxide-induced DNA damage: DNA-bound copper ion primarily induces base modifications

The kinetics of frank DNA strand breaks and DNA base modifications produced by Cu(II)/ascorbate/H2O2 were simultaneously determined in purified human genomic DNA in vitro. Modified bases were determined by cleavage with Escherichia coli enzymes Nth protein (modified pyrimidines) and Fpg protein (modified purines). Single-stranded lesion frequency before (frank strand breaks) and after (modified bases) Nth or Fpg protein digestion was quantified by neutral glyoxal gel electrophoresis. Dialysis of EDTA-treated genomic DNA purified by standard proteinase K digestion/phenol extraction was necessary to remove low molecular weight species, probably transition metal ions and metal ion chelators, which supported frank strand breaks in the presence of ascorbate + H2O2 without supplemental copper ions. We then established a kinetic model of the DNA-damaging reactions caused by Cu(II) + ascorbate + H2O2. The principal new assumption in our model was that DNA base modifications were caused exclusively by DNA-bound Cu(I) and frank strand breaks by non-DNA-bound Cu(I). The model was simulated by computer using published rate constants. The computer simulation quantitatively predicted: (1) the rate of H2O2 degradation, which was measured using an H2O2-sensitive electrode, (2) the linearity of accumulation of DNA strand breaks and modified bases over the reaction period, (3) the rate of modified base accumulation, and (4) the dependence of modified base and frank strand production on initial Cu(II) concentration. The simulation significantly overestimated the rate of frank strand break accumulation, suggesting either that the ultimate oxidizing species that attacks the sugar-phosphate backbone is a less-reactive species than the hydroxyl radical used in the model and/or an unidentified hydroxyl radical-scavenging species was present in the reactions. Our experimental data are consistent with a model of copper ion-DNA interaction in which DNA-bound Cu(I) primarily mediates DNA base modifications and nonbound Cu(I) primarily mediates frank strand break production.

Endogenous lesions, S-phase-independent spontaneous mutations, and evolutionary strategies for base excision repair

We calculate from published levels of endogenous base lesions that our cells constantly generate and excise during base excision repair (BER) about one million lesions per day. Repair glycosylases may also non-specifically excise an additional number of undamaged bases. The resulting abasic sites are repaired daily by BER. The fidelity of polymerase-beta is 2.4x10(-5) and one must postulate additional fidelity mechanisms in the BER complex to explain the low mutation rate of resting cells. Any strategy which constitutively increases glycosylase activity to prevent endogenous lesions from entering S-phase and becoming mutations will also serve to increase the number of mutations per day caused by non-specific excision of normal undamaged bases. The best break-even strategy for reducing endogenous lesion-induced mutations is clearly not one of avid repair. Lower organisms from bacteriophage to fungi have adopted strategies to generate 0.0033 consequential mutations per cell division, no more and no less. Strategies such as down regulating glycosylase activity outside of S-phase to reduce time-dependent mutation frequency while leaving lesion replication-induced mutation frequency unchanged are discussed.

Somatic mutation theory, DNA repair rates, and the molecular epidemiology of p53 mutations

The theory of somatic mutagenesis predicts that the frequency pattern of induced selectable mutations along a gene is the product of the probability patterns of the several sequential steps of mutagenesis, e.g., damage, repair, polymerase misreading, and selection. Together, the variance of these component steps is propagated to generate a mutagens induced mutational spectrum along a gene. The step with the greatest component of variance will drive most of the variability of the mutation frequency along a gene. This most variable step, for UV-induced mutations, is the cyclobutyl pyrimidine dimer repair rate. The repair rate of cyclopyrimidine dimers is quite variable from nucleotide position to nucleotide position and we show that this variation along the p53 gene drives the C-->T transition frequency of non-melanocytic skin tumors. On showing that the kinetics of cyclopyrimidine dimer repair at any one nucleotide position are first order, we use this kinetic and the somatic mutation theory to derive Leq, the adduct frequency along a gene as presented to a DNA polymerase after a cell population reaches damage-repair equilibrium from a chronic dose of mutagen. Leq is the product of the first two sequential steps of mutagenesis, damage and repair, and the frequency of this product is experimentally mapped using ligation-mediated PCR. The concept of Leq is applied to mutagenesis theory, chronic dose genetic toxicology, genome evolution, and the practical problems of molecular epidemiology.

Adaptive enhancement and kinetics of nucleotide excision repair in humans.

An adaptive response, low doses of a mutagen rendering cells more able to subsequently cope with higher doses of that or a related challenging mutagen, enhances nucleotide excision repair in human fibroblasts. After fibroblasts were flashed with 20 J/m2 of UVC, the cyclopyrimidine dimer frequency at any single dinucleotide position remained unchanged for several hours before abruptly displaying first order kinetics of repair. These kinetics were determined by ligation-mediated PCR along exon 9 of the human p53 gene. When a chronic dose of quinacrine mustard (QM) preceded the UVC challenge, the duration of the cyclobutane pyrimidine dimer (CPD) repair lags were reduced by a factor of three and the kinetic half-lives for CPD repair were reduced by a factor of three. The observed repair kinetics are consistent with the following model. The UVC dose required (K(m)) to generate a substrate concentration which half-saturates the cells repair capacity is 3 J/m2 for the high affinity (6-4) photoproducts and greater than 100 J/m2 for the low affinity cyclobutane dimers. After 20 J/m2 of UVC, the repair enzyme is saturated with (6-4) photoproducts; these competitively inhibit CPD repair by binding all available repair enzyme. After the (6-4)s are repaired, the CPD concentration is less than K(m)(CPD) and so CPD repair kinetics initiate with first order kinetics. QM-induced enhancement, by increasing the concentration, Vmax, of repair enzyme, shortens the duration of (6-4) saturation and increases the rate constant for cyclobutane dimer repair. The data exactly fit the expectations from Michaelis kinetics. Transcription coupled repair is less amenable to Michaelis interpretations and enhanced global repair was almost as rapid as the slightly enhanced transcription coupled repair. We infer that repair enhancement is unable to proportionally increase the number of matrix attachment sites necessary for transcription coupled repair. Understanding competitive inhibition between adduct classes and adaptive enhancement of Vmax is important to understanding the effects of high doses of mutagen mixtures.

Sister chromatid exchange and chromosome organization based on a bromodeoxyuridine Giemsa-C-banding technique (TC-Banding)

Hoechst 33258 fluorescent staining of bromodeoxyuridine substituted chromosomes provided a high resolution technique for following the segregation of replicated chromosomal DNA (Latt, 1973). Modifications have produced the same results after Giemsa staining (Wolff and Perry, 1975). Since this does not necessarily require Hoechst (Korenberg and Freedlander, 1975), we call this bromodeoxyuridine-Giemsa banding (BG-banding). We here describe a further modification which allows one to follow the T-rich strand of the AT-rich satellite DNA of C-band heterochromatin. We call this TC-banding. This technique was used to examine metacentric marker chromosomes found in mouse L-cells that contain many interstitial blocks of centromeric-type heterochromatin in each arm plus the usual two blocks of centromeric heterochromatin. One of the advantages of this technique for such chromosomes is that it is possible to distinguish first from second cell cycle sister chromatid exchange and unambiguously detect centromeric sister chromatid exchange. We found some chromosomes to have high rates of centromeric sister chromatid exchange. After one cycle in bromodeoxyuridine we could examine the satellite polarity of the heterochromatic DNA. Since there was no change in satellite polarity in any of the heterochromatic blocks, marker chromosomes could not have been formed by paracentric inversions, inverted insertions or inverted translocations. These results allow the formulation of several rules of chromosome organization.

Organization of mutations along the genome: a prime determinant of genome evolution.

Recent advances in molecular mutagenesis reveal that two of the mechanisms which contribute to mutagen-induced point mutations, the frequency of induced DNA damage and the repair rate of this damage, vary considerably along the genome. At a grosser level of genomic resolution, cytogeneticists now distinguish several classes of chromosome bands along human chromosomes. The hot spots for X-ray induced breaks (chromosome mutations) occur in certain band classes, while the hot spots for mitomycin C-induced exchanges or melphalan-induced breaks occur in other band classes. Knowledge of these mutation patterns is modifying our concepts of genome evolution.

Chromatin self-organization by mutation bias

Proteins, on binding to a DNA sequence, alter the frequency and quality of mutations that occur in the sequence. This represents a reverse flow of information from proteins to DNA. Nucleosome binding causes patterns of UV-induced damage which, when converted to mutations by replication, will phase nucleosomes. We propose that DNA binding proteins create their own high- or low-affinity binding sites along DNA sequences by biased mutational pressure.

Algorithm for writing a scientific manuscript

We present an algorithm for the construction of a strong initial draft. It is designed to overcome writers block and to assist scientists who are not native English speakers. The writing starts with making figures and tables. These suggest several terse summary statements, the few major conclusions or observations the author will present to the scientific community. After identification of the audience, the specific community addressed, materials and methods are written to explain how the tables and figures were generated. Results are initially restricted to describing the logical data relations in each table and figure. The discussion then converts each data relationship into mechanistic cause‐and‐effect interpretations suitable for the abstract. A brief epilogue deals with the submission and the fate of the final manuscript once submitted. Although other models for the initial draft exist, this model has worked for us and new researchers in our laboratories and addresses problems we encountered while editing manuscripts.

DNA replication asynchrony between the paternal and maternal alleles of imprinted genes does not straddle the R/G transition.

Abstract. Imprinted autosomal loci apparently reside in very large chromosomal domains that exhibit asynchrony in replication of homologous alleles during the DNA synthesis phase. Replication asynchrony can be cytogenetically visualized by a replication-banding discordance between homologous bands of a given pair of chromosomal homologs. The replication time of a chromosomal band at high resolution can be determined by blocking DNA synthesis at the R/G-band transition and using replication banding. The R/G transition reflects the transition from early (R-) to late (G- and C-) band DNA replication. We studied discordance between two groups of homologous chromosomal bands: (a) four bands, 6q26–27, 11p13, 11p15.5 and 15q11.2–12, each containing at least one imprinted gene; and (b) nine bands containing no known imprinted genes. Fifty pairs of chromosomes were analyzed at high resolution after R/G transition blocking and late 5-bromo-2′-deoxyuridine incorporation. The rate of discordance was the same for bands containing imprinted genes and for control bands. Both homologous bands of a pair replicate either before or after the R/G transition and do not straddle the R/G transition. Repression associated with imprinting does not appear to involve late replication at the band level of resolution. Tissue-specific inactivation is associated with DNA methylation and late replication, whereas allele-specific inactivation is associated with DNA methylation but not with delayed or late replication.

Chronic low-dose lesion equilibrium along genes: measurement, molecular epidemiology, and theory of the minimal relevant dose

Spontaneous mutations seem to be caused almost entirely by endogenous lesions. The pattern of these lesions along a gene represents an equilibrium between damage and repair. A pattern can be measured using ligation-mediated PCR (polymerase chain reaction) and a large chronic dose of a suspected endogenous mutagen. A study using dimethylsulfate-induced 7meGuanine lesions indicates that the exogenously induced pattern depends on how methyl purine glycosylase recognizes sequence context and, for this lesion, the pattern may be independent of the mutagens dose.