Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.     

Inhibition of homologous recombination by variants of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs)

Two major DNA double-strand break repair pathways exist in all eukaryotes, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Although both pathways can function throughout the cell cycle, NHEJ predominates in G0/G1) (when a replicated sister chromatid is unavailable), whereas HR makes a more substantial contribution in S and G2. How a cell chooses between these two important DNA repair pathways is largely unknown. DNA-dependent protein kinase (DNA-PK) is critical for NHEJ. Here, we describe two conserved splice variants of a catalytic subunit of DNA-PK (DNA-PKcs) that are expressed predominately in nondividing cells. Although both encode stable products, neither reverses the NHEJ defects in DNA-PKcs-deficient cells. In fact, cells expressing one of the DNA-PKcs variants are slightly more radiosensitive than cells completely deficient in DNA-PKcs. We investigated whether cells expressing the DNA-PKcs variants had any other DNA repair deficits and found that these cells are considerably more sensitive to both etoposide and mitomycin C than cells that express no DNA-PKcs at all. Because repair of DNA damage induced by these two agents requires intact HR, we tested whether the NHEJ-defective variants of DNA-PKcs inhibit double-strand break-induced HR in an integrated substrate. In cells expressing the NHEJ-defective variants, HR was markedly reduced. Because the splice variants are expressed highly only in nondividing cells, quiescent cells would be afforded a mechanism to inhibit repair by means of HR when sister chromatids are not available as templates for accurate repair with low risk of genome rearrangement, thereby enhancing genome stability.     

A phage P1 function that stimulates homologous recombination of the Escherichia coli chromosome.

Recombination between two different defective lacZ genes in the Escherichia coli chromosome (lac- X lac- recombination) was stimulated 2- to 8-fold by prophage P1, depending on the nature of the phage c1 repressor. The P1 BamHI restriction fragment B8 in a lambda-P1:B8 hybrid phage, stimulated lac- X lac- recombination 90-fold in the absence of P1 repressor. A gene necessary for recombination enhancement, designated ref, was localized to one end of B8. Ref expression from lambda-P1:B8 was repressed in trans by a P1 c+ prophage. Two P1 regulatory mutations, bof and lxc, derepressed prophage expression of ref and depressed a prophage function that complemented an E. coli mutant (ssb) deficient in the single-stranded DNA binding protein. Ref stimulation was dependent on preexisting E. coli recombination functions (RecA-RecBC and RecA-RecF). However, other (phage and plasmid) recombination processes involving these functions were not stimulated. ref::Tn5 phages plated and formed lysogens normally. Thus ref appears to be an integral, but not essential, phage gene that stimulates recombination of the host chromosome specifically.     

Scaffolding protein SPIDR/KIAA0146 connects the Bloom syndrome helicase with homologous recombination repair

The Bloom syndrome gene product, BLM, is a member of the highly conserved RecQ family. An emerging concept is the BLM helicase collaborates with the homologous recombination (HR) machinery to help avoid undesirable HR events and to achieve a high degree of fidelity during the HR reaction. However, exactly how such coordination occurs in vivo is poorly understood. Here, we identified a protein termed SPIDR (scaffolding protein involved in DNA repair) as the link between BLM and the HR machinery. SPIDR independently interacts with BLM and RAD51 and promotes the formation of a BLM/RAD51-containing complex of biological importance. Consistent with its role as a scaffolding protein for the assembly of BLM and RAD51 foci, cells depleted of SPIDR show increased rate of sister chromatid exchange and defects in HR. Moreover, SPIDR depletion leads to genome instability and causes hypersensitivity to DNA damaging agents. We propose that, through providing a scaffold for the cooperation of BLM and RAD51 in a multifunctional DNA-processing complex, SPIDR not only regulates the efficiency of HR, but also dictates the specific HR pathway.     

Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans-dihydrodiol activation in human lung A549 cells

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.     

Differential cellular expression of the human MSH2 protein in normal and myelodysplastic haematopoiesis

Loss of human MSH2 (hMSH2) protein might be involved in the multistep pathogenesis of haematological malignancies associated with genetic instability. Here, we examine cellular hMSH2 expression in bone marrow samples from 10 haematopoietically normal individuals in comparison with nine patients with myelodysplastic syndrome (MDS) [one refractory anaemia (RA), two RA with ringed sideroblasts (RARS), four RA with excess blasts (RAEB) and two RAEB in transformation (RAEB-T)]. HMSH2 protein was predominantly expressed in myeloblasts and promyelocytes. Blast cells from three patients with RAEB and one with RAEB-T displayed absent or very low hMSH2 expression. As no correlation between hMSH2 expression and chromosomal aberrations was observed, further genetic events seem to be required to induce karyotype instability.     

An action spectrum (290-320 nm) for TNFalpha protein in human skin in vivo suggests that basal-layer epidermal DNA is the chromophore

Terrestrial solar UVB radiation ( approximately 295-320 nm) readily induces cyclobutane pyrimidine dimers (CPDs) in human skin DNA that result in characteristic mutations associated with nonmelanoma skin cancer. The proinflammatory cytokine TNFalpha is important in mouse skin chemical carcinogenesis and is thought to also play a role in UVR-induced skin cancer by its immunomodulatory properties. There is some in vitro evidence that CPDs initiate the production of TNFalpha, and we tested this hypothesis by comparing the wavelength dependence (action spectrum) for TNFalpha protein induction in human skin in vivo with our earlier in vivo action spectra for CPD induction in four different epidermal layers of human skin. Normal volunteers (n = 35) were irradiated with physiologically relevant doses of monochromatic UVB (290-320 nm), and TNFalpha concentration was assessed, by high-sensitivity ELISA, in exudates from skin suction blisters raised 8 h after irradiation. An action spectrum, constructed from the slopes of the dose-response curves at the different wavelengths, showed maximal efficacy at 300 nm. An excellent match was observed for TNFalpha and the CPD action spectrum for cells in the lower basal epidermis. These data strongly suggest that UVB-induced photodamage to DNA in the epidermal basal layer is a major trigger for TNFalpha production. The TNFalpha may originate directly from the keratinocytes in this layer or inflammatory cells that are rapidly recruited into the upper dermis (e.g., neutrophils) as a consequence of DNA photodamage to basal-layer keratinocytes.