Mark T. Muller

Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats

Alternating purine-pyrimidine sequences (RY repeats) demonstrate considerable homology to the consensus sequence for vertebrate topoisomerase II (Spitzner and Muller (1988) Nucleic Acids Res. 16: 1533-1556). This is shown below and positions that can match are underscored. RYRYRYRYRYRYRYRYRY = alternating purine-pyrimidine 18 bp RNYNNCNNGYNGKTNYNY = topoisomerase II consensus sequence (R is purine, Y is pyrimidine, K is G or T.) Topoisomerase II cleavage reactions were performed (in the absence of inhibitors) on a plasmid containing a 54 base RY repeat and the single strong cleavage site mapped to the RY repeat. Analysis of this DNA on sequencing gels showed that the enzyme cleaved a number of sites, all within the 54 base pair RY repeat. Topoisomerase II also made clustered cleavages within other RY repeats that were examined. Quantitative analysis of homology to the consensus sequence, as measured by the match of a site to a matrix of base proportions from the consensus data base (the matrix mean), showed that both the locations and the frequencies of cleavage sites within RY repeats were proportional to homology scores. However, topoisomerase II cleaved RY repeats preferentially in comparison to non-RY sites with similar homology scores. The activity of the enzyme at RY repeats appears to be proportional to the length of the repeat; additionally, GT, AC and AT repeats were better substrates for cleavage than GC repeats.

DNA Damage, Homology-Directed Repair, and DNA Methylation

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%–4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, ~50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2′-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments.

A secreted calreticulin protein in ixodid tick (Amblyomma americanum) saliva

A complementary DNA clone from salivary glands of feeding female Amblyomma americanum ticks has been characterized as encoding calreticulin. Calreticulin, a major endoplasmic reticulum (ER) calcium-binding protein, appears to be secreted in Amblyomma and Dermacentor saliva. Evidence is accummulating that calreticulin performs roles unrelated to calcium storage. Unlike most known calreticulins, tick-secreted calreticulin lacks the ER retention signal, KDEL. This is the first molecular cloning of a specific tick salivary gland protein. The finding of a secreted calreticulin in tick saliva suggests a role for calreticulin in blood feeding through host immunosuppression or antihemostasis.

SUMOylation enhances DNA methyltransferase 1 activity.

DNA methylation regulates gene expression through a complex network of protein-protein and protein-DNA interactions in chromatin. The maintenance methylase, DNMT1 (DNA methyltransferase 1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications. The mechanistic details that explain how DNMT1 catalytic action is directed and regulated in chromatin are important in our overall understanding of gene control. In this work, we show that DNMT1 is modified by SUMOylation and we have mapped these SUMOylation sites by defined mutations. SUMOylated DNMT1 is catalytically active on genomic DNA in vivo and we find that SUMOylation significantly enhances the methylase activity of DNMT1 both in vitro and in chromatin. These data suggest that SUMOylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in somatic cells.

DNA methyltransferase 1-associated protein (DMAP1) is a co-repressor that stimulates DNA methylation globally and locally at sites of double strand break repair.

Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.

Subnuclear distribution of topoisomerase I is linked to ongoing transcription and p53 status

The nonconserved, hydrophilic N-terminal domain of eukaryotic DNA topoisomerase I (topo I) is dispensable for catalytic activity in vitro but essential in vivo. There are at least five putative nuclear localization signals and a nucleolin-binding signal within the first 215 residues of the topo I N-terminal domain. We have investigated physiological functions of the topo I N-terminal domain by fusing it to an enhanced green fluorescent protein (EGFP). The first 170 residues of the N-terminal domain allow efficient import of chimeric proteins into nuclei and nucleoli. The nucleolar localization of this protein does not depend on its interaction with nucleolin, whereas ongoing rDNA transcription clearly is crucial. Immunoprecipitation experiments reveal that the topo I N terminus (topoIN)-EGFP fusion protein associates with the TATA-binding protein in cells. Furthermore, DNA damage results in extensive nuclear redistribution of the topoIN-EGFP chimeric product. The redistribution is also p53-dependent and the N terminus of topo I appears to interact with p53 in vivo. These results show that the topo I localization to the nucleolus is related to the p53 and DNA damage, as well as changes in transcriptional status. Nucleolar release of topo I under conditions of cellular duress may represent an important, antecedent step in tumor cell killing by topoisomerase active agents.

Toward Drug Repurposing in Epigenetics: Olsalazine as a Hypomethylating Compound Active in a Cellular Context

DNA hypomethylating drugs that act on DNA methyltransferase (DNMT) isoforms are promising anticancer agents. By using a well‐characterized live‐cell system to measure DNA methylation revisions (imprints), we characterize olsalazine, an approved anti‐inflammatory drug, as a novel DNA hypomethylating agent. The cell‐based screen used in this work is highly tractable, internally controlled, and well‐suited for a drug repurposing strategy in epigenetics. Olsalazine very closely mimics the action of 5‐aza‐2′‐deoxycytidine, a known hypomethylating drug, with minimal cytotoxicity at the concentrations tested. Olsalazine was identified by a rapid computer‐guided similarity search of a database of approved drugs to a previously identified inhibitor of DNMTs.

Quantitation of eukaryotic topoisomerase I reactivity with DNA. Preferential cleavage of supercoiled DNA

A method has been used to quantitate the reaction between eukaryotic type I DNA topoisomerase and topological forms of DNA. This procedure (Trask, D.K., DiDonato, J.D. and Muller, M.T. (1984) Eur. Mol. Biol. Organ. J. 3, 671-676) measures the efficiency of DNA cleavage and concurrent formation of a covalent enzyme/DNA complex. Eukaryotic type I topoisomerases react preferentially by 5-10-fold with supercoiled DNA. The effect of supercoiling is clearly evident in that both the initial rate and final extent of the reaction is elevated. Because the dissociation rate is much lower than the association rate, it is possible to isolate native topoisomerase/DNA complexes. These complexes are comprised of enzyme molecules which are catalytically active when challenged with a second supercoiled DNA substrate. Collectively, the data support the conclusion that a functional intermediate in the reaction sequence is being detected and that the avian topoisomerase I preferentially cleaves supercoiled DNA.

Reduced activity of topoisomerase II in an Adriamycin-resistant human stomach-adenocarcinoma cell line

Abstract A human stomach-adenocarcinoma cell line (MKN-45) was selected for resistance to Adriamycin by stepwise exposure to increasing concentrations of this agent. The resulting cell line (MKN/ADR) exhibited a high level of cross-resistance to topoisomerase II (topo II)-targeted drugs such as Adriamycin, mitoxantrone, and etoposide but showed no cross-resistance to other chemotherapeutic agents such as cisplatin, carboplatin, 5-fluorouracil, or mitomycin-C. P-glycoprotein encoded by the mdr-1 gene was not overexpressed in the MKN/ADR cell line. The doubling time of the MKN/ADR cell line (2.1 days) increased only slightly as compared with that of the MKN cell line (1.7 days). The patterns of cross-resistance to various chemotherapeutic agents led us to examine the cellular contents of topo II in both the drug-sensitive and the drug-resistant cells. Extractable topo II enzyme activity was 3-fold lower in MKN/ADR cells as compared with the parental MKN cells. Levels of topoisomerase I (topo I) catalytic activity were similar in both wild-type MKN and drug-resistant MKN/ADR cells. Southern-blot analysis of genomic DNA probed with topo IIα or IIβ showed no sign of either gene rearrangement or hypermethylation. Northern-blot analysis revealed that both topo IIα and topo IIβ mRNA transcripts were essentially identical in the MKN and MKN/ADR cells. In contrast, Western-blot analysis revealed an approximately 20-fold lower level of topo IIα in drug-resistant cells as compared with drug-sensitive cells, whereas topo IIβ levels were similar in both lines. Moreover, the amount of in vivo topo IIα-DNA covalent complexes formed in the presence of etoposide was also approximately 20-fold lower in drug-resistant cells. No mutation was detected in the promoter region of the topo IIα gene in resistant cells as compared with sensitive cells. Thus, low levels of topo IIα polypeptide cannot be ascribed to changes in the mRNA levels. Collectively, the data suggest that a quantitative reduction in topo IIα may contribute to the resistance of MKN cells to Adriamycin and other topo II-targeted drugs.

A predictive model for DNA recognition by the herpes simplex virus protein ICP4.

The herpes simplex virus (HSV) type 1 immediate early protein ICP4 is an essential regulatory enzyme that binds DNA directly in order to stimulate or repress gene expression. The degree of transaction is related to the locations and affinities of the ICP4 binding sites. A number of binding sites have been identified; some sites showed obvious homology to one another, and these were called consensus ICP4 binding sites. Other binding sites did not appear to be related, and these were termed non-consensus sites. We hypothesized, however, that a single model could describe all ICP4 binding sites, given the appropriate characterizations of sites. We performed statistical analyses on a set of ICP4 binding sites and found that the bases important for defining binding were located within a 13 base region. Missing contact analyses on several high-affinity binding sites revealed the same 13 base region as important for critical protein-DNA contacts. From these data we derived the consensus sequence RTCGTCNNYNYSG, where R is purine, Y is pyrimidine, S is C or G, and N is any base. In addition, we found that a better profile for ICP4 binding sites involves use of a matrix of base proportions from the binding site data; sites are analyzed by calculating the Matrix Mean score. We show that this Matrix Mean model could accurately predict the locations of novel ICP4 binding sites. Finally, we analyzed the entire HSV-1 genome for potential ICP4 binding sites and speculate about what these results suggest for the role of ICP4 in viral gene regulation.