DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing.

Expanding mitochondrial base editing tools with broad sequence compatibility is of high need for both research and therapeutic purposes. In this study, we identify a DddA homolog from Simiaoa sunii (Ddd_Ss) which can efficiently deaminate cytosine in DC context in double-stranded DNA (dsDNA). We successfully develop Ddd_Ss-derived cytosine base editors (DdCBE_Ss) and introduce mutations at multiple mitochondrial DNA (mtDNA) loci including disease-associated mtDNA mutations in previously inaccessible GC context. Finally, by introducing a single amino acid substitution from Ddd_Ss, we successfully improve the activity and sequence compatibility of DdCBE derived from DddA of Burkholderia cenocepacia (DdCBE_Bc). Our study expands mtDNA editing tool boxes and provides resources for further screening and engineering dsDNA base editors for biological and therapeutic applications.

Engineering and functional analysis of yeast with a monotypic 40S ribosome subunit.

Emerging evidence reveals that ribosomes are not monolithic but dynamic machines with heterogeneous protein compositions that can reshape ribosomal translational abilities and cellular adaptation to environmental changes. Duplications of ribosomal protein (RP) genes are ubiquitous among organisms and are believed to affect cell function through paralog-specific regulation (e.g., by generating heterogeneous ribosomes) and/or gene dose amplification. However, direct evaluations of their impacts on cell function remain elusive due to the highly heterogeneous cellular RP pool. Here, we engineered a yeast with homogeneous 40S RP paralog compositions, designated homo-40S, by deleting the entire set of alternative duplicated genes encoding yeast 40S RP paralogs. Homo-40S displayed mild growth defects along with high sensitivity to the translation inhibitor paromomycin and a significantly increased stop codon readthrough. Moreover, doubling of the remaining RP paralogous genes in homo-40S rescued these phenotypes markedly, although not fully, compared to the wild-type phenotype, indicating that the dose of 40S RP genes together with the heterogeneity of the contents was vital for maintaining normal translational functionalities and growth robustness. Additional experiments revealed that homo-40S improved paromomycin tolerance via acquisition of bypass mutations or evolved to be diploid to generate fast-growing derivatives, highlighting the mutational robustness of engineered yeast to accommodate environmental and genetic changes. In summary, our work demonstrated that duplicated RP paralogs impart robustness and phenotypic plasticity through both gene dose amplification and paralog-specific regulation, paving the way for the direct study of ribosome biology through monotypic ribosomes with a homogeneous composition of specific RP paralogs.

Structural insight into the recognition of acetylated histone H3K56ac mediated by the bromodomain of CREB-binding protein.

The acetylation of lysine 56 of histone H3 (H3K56ac) enhances the binding affinity of histone chaperones to H3-H4 dimers. CREB-binding protein (CBP) possesses a bromodomain that recognizes H3K56 acetylation. CBP also possesses a histone acetyltransferase (HAT) domain, which has been shown to promote H3K56 acetylation of free histones to facilitate delivery of replication-dependent chaperones to acetylated histones for chromatin assembly. However, the mechanism by which the CBP bromodomain recognizes H3K56ac and the context in which such recognition occurs remain elusive. Here, we solved the crystal structure of the CBP bromodomain in complex with an H3K56ac peptide. Our data demonstrate that the CBP bromodomain recognizes H3K56ac with high affinity. Structural and affinity analyses reveal that the CBP bromodomain prefers an aromatic residue at the -2 position and an arginine at the -4 position from the acetyl-lysine, and that the CBP bromodomain selectively recognizes an extended conformation of the H3 αN helix that contains H3K56ac. We also demonstrate that the CBP bromodomain binds to H3K56ac in a recombinant H3-H4 dimer but not in a mono-nucleosome. Our results suggest that the CBP bromodomain selectively recognizes an extended conformation of the K56-acetylated H3 αN region within an H3-H4 dimer, which is expected to facilitate the HAT activity of CBP for subsequent H3K56 acetylation of free histones. DATABASES: Coordinates of the CBP bromodomain in complex with H3K56ac as described in this article have been deposited in the PDB with accession number 5GH9.

[Detection and screening for human cytomegalovirus infection in allogeneic hematopoietic stem cell transplantation recipients by different methods].

The aim of study was to explore the better detection method for cytomegalovirus (CMV) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) recipients and to compare the efficiency of fluorogenic quantitative PCR (FQ-PCR), flow cytometry (FCM) and ELISA. The plasma DNA loading and serum level of IgM antibody against CMV in 214 clinical specimens from 19 allo-HSCT patients were detected by real-time FQ-PCR and ELISA respectively, the pp65 antigen in 118 peripheral blood leukocyte samples were measured by FCM. The results showed that the positive rates of pp65 antigen, IgM antibody and DNA load were 30.85% (58/188), 13.08% (28/214) and 35.51% (76/214) respectively, the coincidence between their sequential detection positive rates and clinical diagnosis were 7/8, 7/8 and 3/8 respectively. There was no statistical significant difference between the positive rate of pp65 antigen and of DNA amount (P > 0.05), and they have manifested relationships (P < 0.05). The positive rate of IgM antibody detected by ELISA was obvious lower than that of DNA quantitated by FQ-PCR and pp65 antigen detected by FCM, but the difference between them showed statistical significance (P < 0.05), Smaller relativity was found between IgM antibody detection and the other two methods (P > 0.05). It is concluded that FQ-PCR and FCM are sensitive, rapid, suitable and reliable methods for monitoring recipient reactive CMV infection of allo-HSCT recipients and are worthy to extensively use for guiding antiviral therapy.