Recognition and repair of UV lesions in loop structures of duplex DNA by DASH-type cryptochrome

Abstract

DNA photolyases and cryptochromes (cry) form a family of flavoproteins that use light energy in the blue/UV-A region for the repair of UV-induced DNA lesions or for signaling, respectively. Very recently, it was shown that members of the DASH cryptochrome subclade repair specifically cyclobutane pyrimidine dimers (CPDs) in UV-damaged single-stranded DNA. Here, we report the crystal structure of Arabidopsis cryptochrome 3 with an in-siturepaired CPD substrate in single-stranded DNA. The structure shows a binding mode similar to that of conventional DNA photolyases. Furthermore, CPD lesions in double-stranded DNA are bound and repaired with similar efficiency as in single-stranded DNA if the CPD lesion is present in a loop structure. Together, these data reveal that DASH cryptochromes catalyze light-driven DNA repair like conventional photolyases but lack an efficient flipping mechanism for interaction with CPD lesions within duplex DNA.Arabidopsis ͉ DNA repair ͉ photolyase C ryptochromes (cry) and DNA photolyases form a unique family of flavoproteins, with members present in all kingdoms of life (1). This family is divided into several subclades according to sequence similarity and function. DNA photolyases are enzymes that repair cytotoxic and mutagenic DNA lesions induced by UV-B, specifically cis-syn cyclobutane pyrimidine dimers (CPDs) or 6-4 pyrimidine-pyrimidone lesions (6-4 photoproduct) by using light energy in the UV-A/blue region (2). The catalytic cofactor of DNA photolyase is flavin adenine dinucleotide (FAD) in its fully reduced form (FADH Ϫ ), and this is present in a U-shaped conformation, as shown in several DNA photolyase structures (3-7). Catalysis involves electron transfer from the excited catalytic cofactor to the UV-B photoproduct, splitting the cyclobutane or oxetane rings, and electron backtransfer to the semireduced FADH°(1). Excitation of FAD is accomplished either by direct photon absorption or by Förster-type energy transfer from an antenna cofactor (1). Despite considerable sequence and structural similarity with DNA photolyases and common cofactor compositions, cryptochromes generally lost repair activity but gained photoreceptor function operating in the same waveband region as DNA photolyases (1). In plants, cryptochromes trigger several developmental processes, such as deetiolation and photoperiodic flower induction, and they entrain the circadian clock (8). In animals such as Drosophila, cryptochromes also function as photoreceptors for light input to the clock (9) and in magnetoreception (10), whereas mammalian cryptochromes are central components of the circadian clock without proven photoreceptor function (11). The more recently discovered subclade of the cryptochrome/ photolyase family, named cry DASH, includes members in plants, cyanobacteria, eubacteria, and vertebrates (12-15). It has been suggested that they represent photoreceptors because they lacked repair activity for CPDs in double-stranded DNA (dsDNA), despite their DNA-binding activity (12, 13). Positive support fo...

Crystal structure of a prokaryotic (6-4) photolyase with an Fe-S cluster and a 6,7-dimethyl-8-ribityllumazine antenna chromophore

“…In this respect, PhrB differs from prototypical CPFs, in which another Arg residue [Arg421 in Drome (6-4) PL] within the region corresponding to the α17-α18 connecting loop in PhrB stabilizes the flip out of the lesion (Fig. 4 C and D) (17,20,26). This Arg is missing in FeS-BCPs.…”

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution

“…All of the amino acids that act in binding of FAD in Arabidopsis Cry3 (20,35) or are essential for hydrogen bonding of MTHF are conserved across the five species, suggesting that Phycomyces CryA might bind FAD and MTHF in a manner similar to that of other members in the cry-DASH subfamily (35,36) (SI Appendix, Fig. S2).…”

“…However, cry-DASH have structural and photochemical properties more similar to photolyases, retain constricted DNA repair activity, and their possible roles in signaling are not yet well established. Cry-DASH were shown to bind and repair CPDs in single-stranded DNA or loop-structured duplex DNA (19,20), but repair of CPDs in duplex DNA by cry-DASH has only been suggested from in vitro studies (e.g., ref. 21).…”

COP1-Mediated Degradation of BBX22/LZF1 Optimizes Seedling Development in Arabidopsis

“…observation that BBX22 regulates chloroplast development (Chang et al, 2008), genes regulated by BBX22 include ELIP1, ELIP2, CRY3/CRYD, and SIG5/SIGE, which encode chloroplast proteins (Yao et al, 2003;Tsunoyama et al, 2004;Heddad et al, 2006;Pokorny et al, 2008). ELIP1 and ELIP2, transiently induced by different qualities of light, encode thylakoid proteins functioning in chlorophyll biosynthesis (Casazza et al, 2005;Rossini et al, 2006).…”