Biochemical Characterization of the DASH‐Type Cryptochrome CryD From Fusarium fujikuroi

Abstract

Proteins from the cryptochrome/photolyase family utilize UV-A, blue or even red light to achieve such diverse functions as repair of DNA lesions by photolyases and signaling by cryptochromes. DASH-type cryptochromes retained the ability to repair cyclobutane pyrimidine dimers (CPDs) in single-stranded DNA regions in vitro. However, most organisms possess conventional CPD photolyases responsible for repair of these lesions in vivo. Recent work showed that the DASH-type cryptochrome CryD plays a regulatory role in diverse light-dependent processes in Fusarium fujikuroi. Here, we report our in vitro studies on heterologously expressed FfCryD. The purified protein contains N(5) ,N(10) -methenyltetrahydrofolate and flavin adenine dinucleotide as cofactors. Photoreduction and DNA photorepair experiments confirmed that FfCryD is active in light-driven electron transfer processes. However, the protein showed comparable affinities for CPD-comprising and undamaged DNA probes. Surprisingly, after purification, full-length FfCryD as well as a truncated version containing only the PHR domain bound RNA which influenced their behavior in vitro. Moreover, binding of FfCryD to RNA indicates a putative role in RNA metabolism or in posttranscriptional control of gene expression.

The Two Cryptochrome/Photolyase Family Proteins Fulfill Distinct Roles in DNA Photorepair and Regulation of Conidiation in the Gray Mold Fungus Botrytis cinerea

“…Such mechanisms have been suggested previously for F. fujikuroi CryD and N. crassa CRY (23,25). In this context, it is noteworthy that biochemical analyses revealed the ability of fungal cry-DASH proteins to directly bind nucleic acids (23,25,44). For instance, F. fujikuroi CryD is capable of binding RNA at the photolyase domain independently of the putative RNA-interacting R/G-rich region.…”

“…Further sequence analyses predicted a nuclear localization signal ( 192 KRKR 195 ) in the DNA photolyase domain. BcCRY2 also features DNA photolyase (PF00875) (1.6eϪ20) and FAD-binding (PF03441) (3.1eϪ43) domains but contains an additional C-terminal R/G-rich region that is often associated with the ability to interact with RNA (23,(43)(44)(45).…”

“…Since BcCRY1 and BcCRY2 represent the only proteins containing photolyase domains, we consider BcCRY1 the main photolyase of B. cinerea, although a DNA repair activity of BcCRY2 cannot be excluded without biochemical analyses. For instance, the F. fujikuroi cry-DASH does possess DNA repair activity in vitro, but its disruption does not affect photoreactivation (44). In contrast to its ortholog A. nidulans CryA, which acts as a repressor of sexual development (22), BcCRY1 has no obvious regulatory function.…”

“…1C) may indicate a posttranscriptional regulation of conidiation-related genes. In this context, the reported RNA-binding properties (23,44) and the conserved putative RNA-interacting R/G-rich region of fungal cry-DASHs are of special interest. To test whether the C-terminal region is essential for the functionality of the protein, a variant lacking the R/G-rich region (BcCRY2 Δ562-623aa ) (see Fig.…”

Two Photolyases Repair Distinct DNA Lesions and Reactivate UVB-Inactivated Conidia of an Insect Mycopathogen under Visible Light

“…The results indicated a more prominent role of Phr1 than of Phr2 but little role of CryD in photoreactivation of UVB-inactivated conidia. Previously, cry-DASH homologs in bacteria, fungi, plants, and animals were shown to repair CPD lesions of single-stranded DNAs in vitro (36)(37)(38)(39). Since fungal DNA is double-stranded, the cryD mutants showing little change in photoreactivation efficiency were not included in the experiments relating to double-stranded DNA (dsDNA) lesions in vivo.…”