Biomedical subjects

Ismail Hassan Ismail

Publications and source records attributed to Ismail Hassan Ismail.

5 recordsLinked to original sources

PubMed2025

Pathogenic variants in MAEA disrupt DNA replication fork stability and are associated with developmental abnormalities in humans.

Replication stress (RS) poses a threat to genome stability and drives genomic rearrangements. The homologous recombination (HR) pathway repairs stalled replication forks (RFs) and prevents such instability. Through an E3 ubiquitin ligase screen aimed at identifying regulators of RAD51, we identified macrophage erythroblast attacher (MAEA), a core component of C-terminal to Lish (CTLH) E3 ubiquitin ligase complex, as a regulator of the HR pathway. Loss of MAEA impairs RAD51 recruitment at stalled RFs, leading to increased sensitivity to RS-inducing agents and excessive degradation of nascent DNA strands. Mechanistically, MAEA associates with and mediates the ubiquitylation of Ku80, enabling its removal from RF ends and facilitating the loading of RAD51. Notably, MAEA deficiency is associated with a developmental disorder involving microcephaly, craniofacial abnormalities, ocular defects, and heart malformations. Functional assays show that disease-linked MAEA variants (R34C, E349G, Y394D, and M396R) are defective in RS response. These findings establish MAEA as an essential factor in RF protection and genome integrity.

Humans↗

PubMed2004

Activation of ataxia telangiectasia mutated by DNA strand break-inducing agents correlates closely with the number of DNA double strand breaks.

The protein kinase ataxia telangiectasia mutated (ATM) is activated when cells are exposed to ionizing radiation (IR). It has been assumed that ATM is specifically activated by the few induced DNA double strand breaks (DSBs), although little direct evidence for this assumption has been presented. DSBs constitute only a few percent of the IR-induced DNA damage, whereas the more frequent single strand DNA breaks (SSBs) and base damage account for over 98% of the overall DNA damage. It is therefore unclear whether DSBs are the only IR-induced DNA lesions that activate ATM. To test directly whether or not DSBs are responsible for ATM activation, we exposed cells to drugs and radiation that produce different numbers of DSBs and SSBs. We determined the resulting ATM activation by measuring the amount of phosphorylated Chk2 and the numbers of SSBs and DSBs in the same cells after short incubation periods. We found a strong correlation between the number of DSBs and ATM activation but no correlation with the number of SSBs. In fact, hydrogen peroxide, which, similar to IR, induces DNA damage through hydroxyl radicals but fails to induce DSBs, did not activate ATM. In contrast, we found that calicheamicin-induced strand breaks activated ATM more efficiently than IR and that ATM activation correlated with the relative DSB induction by these agents. Our data indicate that ATM is specifically activated by IR-induced DSBs, with little or no contribution from SSBs and other types of DNA damage. These findings have implications for how ATM might recognize DSBs in cells.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

SU11752 inhibits the DNA-dependent protein kinase and DNA double-strand break repair resulting in ionizing radiation sensitization.

Loss of the DNA-dependent protein kinase (DNA-PK) results in increased sensitivity to ionizing radiation due to inefficient repair of DNA double-strand breaks. Overexpression of DNA-PK in tumor cells conversely results in resistance to ionizing radiation. It is therefore possible that inhibition of DNA-PK will enhance the preferential killing of tumor cells by radiotherapy. Available inhibitors of DNA-PK, like wortmannin, are cytotoxic and stop the cell cycle because they inhibit phoshatidylinositol-3-kinases at 100-fold lower concentrations required to inhibit DNA-PK. In an effort to develop a specific DNA-PK inhibitor, we have characterized SU11752, from a three-substituted indolin-2-ones library. SU11752 and wortmannin were equally potent inhibitors of DNA-PK. In contrast, inhibition of the phoshatidylinositol-3-kinase p110gamma required 500-fold higher concentration of SU11752. Thus, SU11752 was a more selective inhibitor of DNA-PK than wortmannin. Inhibition kinetics and a direct assay for ATP binding showed that SU11752 inhibited DNA-PK by competing with ATP. SU11752 inhibited DNA double-strand break repair in cells and gave rise to a five-fold sensitization to ionizing radiation. At concentrations of SU11752 that inhibited DNA repair, cell cycle progression was still normal and ATM kinase activity was not inhibited. We conclude that SU11752 defines a new class of drugs that may serve as a starting point for the development of specific DNA-PK inhibitors.

Adenosine Triphosphate↗

PubMed2004

Determination of protolytic constants by trilinear fluorescence spectroscopy.

Protolytic equilibria often have profound effects on chemical activity, since protolytic species usually behave quite differently. It is therefore important to characterize the protolytic properties of important chemicals. Here we present a new approach to study protolytic equilibria of fluorescent species that is extremely accurate and relies on minimum assumptions. We show that by measuring 2-dimensional excitation/emission scans of samples at different pH. the 3-dimensional experimental data set, I(lambda(ex), lambda(em), C(pH)), can be unambiguously decomposed into the spectral responses of the protolytic species present as well as their concentration. The approach is demonstrated on the protolytic equilibrium of fluorescein. Although the fluorescein monoanion cannot be obtained in pure form, the spectra and concentrations of both fluorescein species, as well as the protolytic constant, are determined with excellent accuracy. The proposed method is general and can be applied not only for studies of protolytic equilibria, but on any chemical equilibria and chemical reactions involving fluorescent species.

Fluorescein↗

PubMed2003

Cleavage of cellular DNA by calicheamicin gamma1.

It is assumed that the efficient antitumor activity of calicheamicin gamma1 is mediated by its ability to introduce DNA double-strand breaks in cellular DNA. To test this assumption we have compared calicheamicin gamma1-mediated cleavage of cellular DNA and purified plasmid DNA. Cleavage of purified plasmid DNA was not inhibited by excess tRNA or protein indicating that calicheamicin gamma1 specifically targets DNA. Cleavage of plasmid DNA was not affected by incubation temperature. In contrast, cleavage of cellular DNA was 45-fold less efficient at 0 degrees C as compared to 37 degrees due to poor cell permeability at low temperatures. The ratio of DNA double-strand breaks (DSB) to single-stranded breaks (SSB) in cellular DNA was 1:3, close to the 1:2 ratio observed when calicheamicin gamma1 cleaved purified plasmid DNA. DNA strand breaks introduced by calicheamicin gamma1 were evenly distributed in the cell population as measured by the comet assay. Calicheamicin gamma1-induced DSBs were repaired slowly but completely and resulted in high levels of H2AX phosphorylation and efficient cell cycle arrest. In addition, the DSB-repair deficient cell line Mo59J was hyper sensitive to calicheamicin gamma. The data indicate that DSBs is the crucial damage after calicheamicin gamma1 and that calicheamicin gamma1-induced DSBs are recognized normally. The high DSB:SSB ratio, specificity for DNA and the even damage distribution makes calicheamicin gamma1 a superior drug for studies of the DSB-response and emphasizes its usefulness in treatment of malignant disease.

Aminoglycosides↗