Biology subjects

Kuska, M.

Publications and source records attributed to Kuska, M..

3 recordsLinked to original sources

bioRxiv2023

Type II restriction of 2-aminoadenine (dZ) modified DNA and production of dZ-modified plasmid in E. coli

The modified DNA base 2,6 aminopurine (2-aminoadenine, (d)Z base) was originally found in phages to counteract host encoded restriction systems. However, only a limited number of restriction endonucleases (REases) have been tested on dZ-modified DNA. Herein we report the results of 147 REases activity on dZ-modified PCR DNA. Among the enzymes tested, 53.1% are resistant or partially resistant, and 46.9% are sensitive when the restriction sites contain 1 to 6 modified bases. Sites with 4-6 dZ substitutions are most likely resistant to Type II restriction. Our results support the notion that dZ-modified phage genomes are evolved to combat host- encoded restriction systems. dZ-modified DNA can also "slow down" phage T5 exonuclease degradation, but it has no effect on RecBCD digestion. When two genes for dZ biosynthesis and one gene for dATP hydrolysis from Salmonella phage PMBT28 (purZ (adenylosuccinate synthetase), datZ (dATP triphosphohydrolase), and mazZ ((d)GTP-specific diphosphohydrolase) were cloned into E. coli plasmid, dZ incorporation level reached 19-20% dZ/(dZ+dA). dZ level can be further increased to 28.9-44.3% with co-expression of a DNA polymerase gene from the same phage. High level of dZ incorporation in recombinant plasmid is possible by co-expression of purZ, mazZ, datZ and phage DNA helicase, dpoZ (DNA polymerase) and ssb (single-stranded DNA binding protein SSB). This work has a general interest for molecular biologists working on dZ DNA modification and restriction systems. It provides a foundation for future research on screening dZ-dependent Type IV restriction systems. The results presented herein may have implication in gene therapy utilizing dZ-modified DNA, provided that human RNA polymerase variants can efficiently perform transcription from a dZ-modified template.

microbiology↗

bioRxiv2023

DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity

J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8, that drives self-assembly through {nu}-{nu} stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity. HIGHLIGHTSDNAJB8 oligomerization is mediated by a short phenylalanine-based motif in the S/T domain Mutation of a single phenylalanine yields a monomeric form of DNAJB8 Monomeric DNABJ8 binds to an aggregation-prone substrate Monomeric DNAJB8 retains substrate aggregation prevention activity

biophysics↗

bioRxiv2022

Universal functions of prion candidates across all three domains of life suggest a primeval role of protein self-templating.

Amyloid-based prions have simple structures, a wide phylogenetic distribution, and a plethora of functions in contemporary organisms, suggesting they may be an ancient phenomenon. However, this hypothesis has yet to be addressed with a systematic, computational, and experimental approach. Here we present a framework to help guide future experimental verification of candidate prions with conserved functions in order to understand their role in the early stages of evolution and potentially in the origins of life. We identified candidate prions in all high-quality proteomes available in UniProt computationally, assessed their phylogenomic distributions, and analyzed candidate-prion functional annotations. Of the 27,980,560 proteins scanned, 228,561 were identified as candidate prions ([~]0.82%). Among these candidates, there were 84 Gene Ontology (GO) terms conserved across the 3 domains of life. We found that candidate prions with a possible role in adaptation were particularly well-represented within this group. We discuss unifying features of candidate prions to elucidate the primeval roles of prions and their associated functions. Candidate prions annotated as transcription factors, DNA binding, and kinases are particularly well suited to generating diverse responses to changes in their environment and could allow for adaptation and population expansion into more diverse environments. We hypothesized that these functions could be evolutionarily ancient, even if individual prion domains themselves are not evolutionarily conserved. Candidate prions annotated with these universally-occurring functions potentially represent the oldest extant prions on Earth and are therefore excellent experimental targets.

bioinformatics↗