Cryptochromes (CRY) are light-responsive flavoproteins that play central roles in nature and human health, including circadian rhythm regulation. They are closely related to photolyases (PL), but, unlike PL, they cannot repair cyclobutane pyrimidine dinners (CPD) or [6-4] photoproducts in duplex DNA. Yet, if the barrier for flipping the CPD from the duplex is reduced, CRY-DASH, the subclass most structurally homologous to CPD PL, binds and repairs CPD like PL. Here, using limited proteolysis, we have identified the most flexible loops in CPD PL. One corresponds to a "recognition loop" that changes conformation substantially during Substrate binding, and engages key interactions with the flipped CPD and the complementary DNA Strand. Proteolysis kinetics reveal that the homologous loop in CRY-DASH is at least 10-fold more reactive. We propose that heightened dynamics of the recognition loop in CRY-DASH contribute to its compromised DNA base flipping, and its evolution of divergent function from PL. (C) 2009 Elsevier Inc. All rights reserved.