Center for Molecular & Translational Medicine

5-Methylcytosine (5mC), the major modified DNA base in mammalian cells, can be oxidized enzymatically to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the Ten-Eleven-Translocation (TET) family of proteins. Whereas 5fC and 5caC are recognized and removed by base excision repair proteins, the 5hmC base accumulates to substantial levels in certain cell types such as brain-derived neurons and is viewed as a relatively stable DNA base. As such, the existence of "reader" proteins that recognize 5hmC would be a logical assumption, and various searches have been undertaken to identify proteins that specifically bind to 5hmC and the other oxidized 5mC bases. However, the existence of definitive 5hmC "readers" has remained unclear and proteins interacting specifically with 5fC or 5caC are also very few. On the other hand, 5hmC is incapable of interacting with a number of proteins that recognize 5mC at CpG sequences, suggesting that 5hmC is an anti-reader modification that may serve to displace 5mC readers from DNA. In this review article, we discuss candidate proteins that may interact with oxidized 5mC bases.

Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment.

The RNA binding domain abundant in apicomplexans (RAP) is a protein domain identified in a diverse group of proteins, called RAP proteins, many of which have been shown to be involved in RNA binding. To understand the expansion and potential function of the RAP proteins, we conducted a hidden Markov model based screen among the proteomes of 54 eukaryotes, 17 bacteria and 12 archaea. We demonstrated that the domain is present in closely and distantly related organisms with particular expansions in Alveolata and Chlorophyta, and are not unique to Apicomplexa as previously believed. All RAP proteins identified can be decomposed into two parts. In the N-terminal region, the presence of variable helical repeats seems to participate in the specific targeting of diverse RNAs, while the RAP domain is mostly identified in the C-terminal region and is highly conserved across the different phylogenetic groups studied. Several conserved residues defining the signature motif could be crucial to ensure the function(s) of the RAP proteins. Modelling of RAP domains in apicomplexan parasites confirmed an ⍺/β structure of a restriction endonuclease-like fold. The phylogenetic trees generated from multiple alignment of RAP domains and full-length proteins from various distantly related eukaryotes indicated a complex evolutionary history of this family. We further discuss these results to assess the potential function of this protein family in apicomplexan parasites.