UV irradiation induces the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts in DNA. These two types of lesions can be directly photorepaired by CPD photolyases and 6-4 photolyases, respectively. Recently, a new class of 6-4 photolyases named iron–sulfur bacterial cryptochromes and photolyases (FeS-BCPs) were found, which were considered as the ancestors of all photolyases and their homologs—cryptochromes. However, a controversy exists regarding 6-4 photoproducts only constituting ∼10–30% of the total UV-induced lesions that primordial organisms would hardly survive without a CPD repair enzyme. By extensive phylogenetic analyses, we identified a novel class of proteins, all from eubacteria. They have relatively high similarity to class I/III CPD photolyases, especially in the putative substrate-binding and FAD-binding regions. However, these proteins are shorter, and they lack the “N-terminal α/β domain” of normal photolyases. Therefore, we named them short photolyase-like. Nevertheless, similar to FeS-BCPs, some of short photolyase-likes also contain four conserved cysteines, which may also coordinate an iron–sulfur cluster as FeS-BCPs. A member from Rhodococcus fascians was cloned and expressed. It was demonstrated that the protein contains a FAD cofactor and an iron–sulfur cluster, and has CPD repair activity. It was speculated that this novel class of photolyases may be the real ancestors of the cryptochrome/photolyase family.

The partitioning of nucleic acids is sensitive to pH during phenol extraction. However, the exact effects of pH on phenol extraction had not been systematically investigated, and the mechanism of which were not fully elucidated. In this paper, we showed that the partitioning of nucleic acids was determined neither solely by the pH of the aqueous buffer being used, nor by the “pH of the phenol”; the latter is a completely wrong conception. We demonstrated that a key determinant for nucleic acid partitioning during phenol extraction was the equilibrated pH of the aqueous phase, which should be defined as the pH of phenol extraction. For example, when 50 mM NaAc-HAc buffer at pH of 3.47 was mixed with an equal volume of water-saturated phenol, the equilibrated pH of aqueous phase would be raised to ∼3.84. At this pH, almost all of genomic DNA partitioned into the phenol phase, and genomic DNA-free total RNA was retained in the aqueous phase. Several salts were found affecting the partitioning of nucleic acids during phenol extraction in different manners. Based on these results, a low-cost and efficient method for genomic DNA-free total RNA extraction was developed.

Synechococcus elongatus, formerly known as Anacystis nidulans, is a representative species of cyanobacteria. It is also a model organism for the study of photoreactivation, which can be fully photoreactivated even after receiving high UV doses. However, for a long time, only one photolyase was found in S. elongatus that is only able to photorepair UV induced cyclobutane pyrimidine dimers (CPDs) in DNA. Here, we characterize another photolyase in S. elongatus, which belongs to iron-sulfur bacterial cryptochromes and photolyases (FeS-BCP), a subtype of prokaryotic 6–4 photolyases. This photolyase was named SePhrB that could efficiently photorepair 6–4 photoproducts in DNA. Chemical analyses revealed that SePhrB contains a catalytic FAD cofactor and an iron-sulfur cluster. All of previously reported FeS-BCPs contain 6,7-dimethyl-8-ribityllumazine (DMRL) as their antenna chromophores. Here, we first demonstrated that SePhrB possesses 7,8-didemethyl-8-hydroxy-5-deazariboflavin (8-HDF) as an antenna chromophore. Nevertheless, SePhrB could be photoreduced without external electron donors. After being photoreduced, the reduced FAD cofactor in SePhrB was extremely stable against air oxidation. These results suggest that FeS-BCPs are more diverse than expected which deserve further investigation.