Biology subjects

Brothers, M.

Publications and source records attributed to Brothers, M..

3 recordsLinked to original sources

bioRxiv2025

Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing owing to their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems such as Cas12a and SpCas9. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here, we characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Cas12f-MG119-28, which supports robust genome editing in human cells. Through structural, biochemical, and kinetic analyses, we compare Cas12f-MG119-28 with two recently described orthologs, Oscillibacter sp. Cas12f (OsCas12f) and Ruminiclostridium herbifermentans Cas12f (RhCas12f). These orthologs present divergent architectures and regulatory features governing PAM recognition, gRNA binding, dimerization, and DNA cleavage. Notably, Cas12f-MG119-28 achieves efficient R-loop formation via a stable dimer interface and a naturally optimized guide RNA. These discoveries elucidate key mechanistic determinants of Cas12f activity and may offer a framework for engineering compact genome editors with therapeutic potential.

biochemistry↗

bioRxiv2021

Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: Recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single base-pair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the non-site-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah{Delta}, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3 overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

molecular biology↗

bioRxiv2019

POL30 alleles in Saccharomyces cerevisiae reveal complexities of the cell cycle and ploidy on heterochromatin assembly

In Saccharomyces cerevisiae, transcriptional silencing at HML and HMR maintains mating-type identity. The repressive chromatin structure at these loci is replicated every cell cycle and must be re-established quickly to prevent transcription of the genes at these loci. Mutations in a component of the replisome, the Proliferating Cell Nuclear Antigen (PCNA), encoded by POL30, cause a loss of transcriptional silencing at HMR. We used an assay that captures transient losses of silencing at HML and HMR to perform extended genetic analyses of the pol30-6, pol30-8, and pol30-79 alleles. All three alleles destabilized silencing only transiently and only in cycling cells. Whereas pol30-8 caused loss of silencing by disrupting the function of Chromatin Assembly Factor 1 (CAF-I), pol30-6 and pol30-79 acted through a separate genetic pathway but one still dependent on histone chaperones. Surprisingly, the silencing-loss phenotypes depended on ploidy but not on POL30 dosage or mating-type identity. Separately from silencing loss, the pol30-6 and pol30-79 alleles also displayed high levels of mitotic recombination in diploids. These results established that histone trafficking involving PCNA at replication forks is crucial to the maintenance of chromatin state and genome stability during DNA replication. They also raised the possibility that increased ploidy may protect chromatin states when the replisome is perturbed.

genetics↗