DNA Repair

Demosponges have an efficient DNA excision repair system. They are able to repair DNA damage caused by genotoxins, e.g. benzo[a]pyrene (Zahn et al. 1983). The mechanisms of repair in sponges are similar to those found in higher Metazoa. We found that in G. cydonium the expression of a homologue of the human XPB/ERCC-3 excision repair gene is induced after exposure to UV radiation (Batel et al. 1998). In humans, the product of the XPB/ERCC-3 gene is involved in the early step of DNA excision repair (Sancar 1996a); it corrects the repair defect in xeroderma pigmentosum and in Cockayne's syndrome. The human XPB/ERCC-3 gene product is a helicase unwinding the DNA in the direction from the site of the lesion (Weeda et al. 1991). Laboratory studies revealed that after irradiation of G. cydonium with 30 or 100 mJ/cm2 UV-B light, a dose-dependent increase in the steady-state level of GCXPB occurs; values of up to 29-fold with respect to the controls which were kept in the dark have been determined. In parallel, the DNA integrity in the sponge samples was measured using Fast Micromethod. The data revealed that the degree of DNA strand breaks paralleled the increase in expression of the GCXPB gene (Batel et al. 1998).

(6-4) Photolyase

Sponges have not only an excision DNA repair system, but also a photolyase-based photoreactivating system. Two types of DNA photolyases have been found that cleave cyclobutane pyrimidine dimers (CPD): CPD photolyase and (6-4) photolyase (Sancar 1996b). The photolyases are part of the cryptochrome family and associated with the blue light photoreceptors (Kanai et al. 1997; Kobayashi et al. 2000). The photolyase of the hexactinellid sponge Aphrocallistes vastus was studied (Schröder et al. 2003). A cDNA was isolated from A. vastus, which comprises high sequence similarity to genes encoding the protostomian and deuterostomian (6-4) photolyases. The A. vastus sequence was assigned to the class I photolyases based on its high sequence similarity, especially within the N-terminal a/ß-domain and C-terminal helical domain. Using functional studies, we demonstrated that this gene codes for a photolyase-related protein. After transfection into the Escherichia coli SY2 strain, which is deficient in photoreversal activity for pyrimidine cyclobutane dimers (Kim and Sundin 2001), the sponge gene caused resistance of the bacteria to UV light (Fig. 10). Irradiation was performed with a UV-B lamp (peak at 312 nm). The UV-induced damage in the bacteria was almost completely repaired during the light-repair phase; at a dose of 0.2 J/m2 only a small reduction in the survival rate was seen (Fig. 10). A strong UV-B sensitivity was observed if the cells were transformed with the empty vector,

Fig. 10. Survival of E. coli strain SY2/pGEX (which does not have photolyase activity; open circles),E. coli SY2/pMS969 [complemented with the ph (6-4) photolyase gene from E. coli; solid squares), and E. coli SY2 carrying the A. vastus APHVAPH gene (solid triangles) after UV-B irradiation at doses between 0.5 and 2 J/m2. Following UV irradiation, photoreacti-vation was allowed to occur by irradiation of the bacteria for 1 h with a lamp emitting visible light (Translux EC halogen photocuring unit, 400 and 520 nm with a maximum at 480 nm; Heraeus Kulzer, Wehheim, Germany). Subsequently, the bacteria were incubated overnight at 26 °C. Survival rate, which is given in percent, was calculated on the basis of number of colonies formed. Mean ± SE (n=5)

irrespective of a post-treatment with light. The recombinant sponge protein bound to UV-modified DNA that contained thymine dimers, while it failed to bind to non-treated DNA, suggesting that the sponge gene displays thymidine dimer-repairing enzyme activity (Schröder et al. 2003).

The detrimental effects of increasing solar UV radiation on corals have been recognized for a long time (Jokiel 1980; Shick et al. 1996). Exposure to UV light has been implicated in the process of coral bleaching. In addition, UV-induced DNA damage in coral-reef microbial communities has been observed (Lyons et al. 1998). As a result of the increased UV exposure, a decrease in skeletal growth and an increase in larval production have been described (Jokiel and York 1982). Most of these studies did not distinguish between the effects of UV-A and UV-B, although the latter wavelengths are more affected by stratospheric ozone depletion.

Primmorphs from Dendronephthya klunzingeri were used to determine the effect of increasing exposure to UV-B and visible light (480 nm). The modified procedure was used to dissociate the coral cells and form primmorphs (Custodio et al. 1998; Müller et al. 1999). The results revealed that primmorphs irradiated with UV-B responded with an increased expression of HSP90 only if exposed to low levels of UV-B (Wiens et al. 2000; Ammar et al. 2001). After irradiation with 30 J/cm2 of UV-B, the primmorphs reacted with a 5.5-fold increase in HSP90 protein, compared with the controls, as revealed in Western blot experiments. Upregulation was observed following an increase in UV-B irradiation to 100 J/cm2, while at levels above 100 J/cm2 no

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