Baculoviral and marsupial CPD photolyases: DNA repair proteins with a circadian clock function

Baculoviruses infect insects and are highly virulent, host specific and environmentally safe, and therefore, are used as biocontrol agents of pest insects. Their effective use in the field is hampered, however, by the ultraviolet (UV) light, which induces cyclobutane pyrimidine dimers (CPDs) in (viral) DNA. CPD photolyases are enzymes that repair CPDs with the help of visible light in a process called photoreactivation. The baculovirus Chrysodeixis chalcites nucleoplyhedrovirus (ChchNPV) possesses two photolyase genes, Cc-phr1 and Cc-phr2. Only Cc-phr2 encodes an enzymatically active photolyase. CPD photolyases are members of the cryptochrome/photolyase family (CPF), which consists of two types of proteins that are structurally conserved, but have different functions. Whereas photolyases repair DNA, cryptochromes work as photoreceptors or regulators of the circadian clock. In mammals, cryptochromes act as inhibitors of CLOCK/BMAL1-driven transcription, and hence, regulate proper functioning of the 24h oscillator. This thesis focuses on the origin and function of baculovirus CPD photolyases. Phylogenetic analysis indicated that a single horizontal gene transfer from an ancestral lepidopteran host led to the current presence of phr genes in a particular clade of the family Baculoviridae. A next study examined whether ChchNPV occlusion bodies (OBs) profited from the photolyases in becoming less UV sensitive. However, this was not the case, as the OBs’s DNA could not be photoreactivated after UV irradiation. This led to the hypothesis that Cc-PHRs may have another function in baculovirus pathogenesis, or alternatively, may induce behavioral changes in the host. Considering the homology to cryptochromes, a possible role of CPD photolyases in the molecular oscillator (‘clock’) was studied. This revealed that Cc-PHR2 and the Potorous tridactylus photolyase (PtCPD-PL), in contrast to Cc-PHR1 and Arabidopsis thaliana (6-4) photolyase, were able to substitute for mammalian cryptochromes. Both Cc-PHR2 and PtCPD-PL inhibited CLOCK/BMAL1-driven transcription and dampened the oscillation of cultured fibroblasts, probably due to the observed interaction with the CLOCK protein. Moreover, both CPD photolyases revived oscillations in arrhythmic cryptochrome knockout (Cry1-/-/Cry2-/-) mouse dermal fibroblasts and livers, showing that Cc-PHR2 and PtCPD-PL can work as true cryptochromes. Chromatin- and co-immunoprecipitation experiments showed that Cc-PHR2 and PtCPD-PL do not prevent CLOCK to bind chromatin nor do they disrupt CLOCK/BMAL1 heterodimer formation. Therefore, these proteins probably use a similar mechanism as mammalian cryptochromes to drive the clock and their overall structure rather than their amino acid sequence and/or domain availability determines their functionality. The gathered data advanced our understanding of the functional evolution of the CPF and showed that the studied class II CPD photolyases are dually functional proteins that repair DNA, but can also regulate the circadian clock in a similar manner as cryptochromes.

Key words: photolyases, cryptochromes, circadian clock, DNA repair, functional evolution, baculoviruses