Biology subjects

Myllykallio, H.

Publications and source records attributed to Myllykallio, H..

6 recordsLinked to original sources

bioRxiv2025

Genome-wide ribonucleotide detection in Archaea

Genome integrity is constantly challenged by the incorporation of ribonucleotides (rNMPs) during DNA synthesis. Covalently linked single and several consecutive rNMPs occur in the genome of a number of organisms. They are mainly introduced by DNA polymerases during DNA replication and repair. In general, cells evolved ribonucleases H (RNases H) specialized in the removal of rNMPs from DNA to avoid any detrimental consequences on genome stability. Here, we describe the involvement of types 1 and/or 2 RNases H in processing embedded rNMPs in the genome of two archaeal species Haloferax volcanii and Thermococcus barophilus. Using combined approaches that include alkaline DNA fragmentation, high-throughput ribose-seq DNA sequencing and nucleotide pool quantification, the distribution, identity, level and sequence context of genomic rNMPs are reported and discussed regards to the intracellular balances of dNTPs and rNTPs. Our results confirm the predominant role of type 2 RNase H in the removal of genomic rNMPs. They also reveal rNMP-base compositions, densities, locations, and variations of surrounding bases at rNMP-embedment for each mutant. The cellular roles of the different RNases H in processing rNMPs in the genome of Archaea are discussed.

genomics↗

bioRxiv2025

Genome wide analyses reveal the role of mutator phenotypes in the emergence of drug resistance in Mycobacterium tuberculosis

Antimicrobial combination therapy is widely used to combat Mycobacterium tuberculosis (Mtb), yet resistance rates continue to rise. Mutator strains, with defects in DNA repair genes, are a known source of resistance in other bacterial infections, but their global role in Mtb remains unclear. Here, we provide a comprehensive overview of the contribution of single nucleotide polymorphisms (SNPs) in DNA Repair, Replication, and Recombination (3R) genes to resistance in Mtb. Through large-scale bioinformatics analysis of 53,589 whole-genomes, we identified 18 novel SNPs linked to genotypic drug resistance in 3R genes, covering 12.5% of clinical isolates/strains with available genome sequences. Notably, a number of the detected SNPs were positively selected during Mtb evolution. Experimental mutation frequency tests further indicated functional defects of these sequence variants in key DNA repair pathways. Our findings indicate that the role of mutability in 3R genes plays a significant role in Mtb resistance at the global level.

evolutionary biology↗

bioRxiv2023

Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS

The linear chromosome of Streptomyces exhibits a highly compartmentalized structure with a conserved central region flanked by variable arms. As double strand break (DSB) repair mechanisms play a crucial role in shaping the genome plasticity of Streptomyces, we investigated the role of EndoMS/NucS, a recently characterized endonuclease involved in a non-canonical Mismatch Repair (MMR) mechanism in archaea and actinobacteria, that singularly corrects mismatches by creating a DSB. We showed that Streptomyces mutants lacking NucS display a marked colonial phenotype and a drastic increase in spontaneous mutation rate. In vitro biochemical assays revealed that NucS cooperates with the replication clamp to efficiently cleave G/T, G/G and T/T mismatched DNA by producing DSBs. These findings are consistent with the transition-shifted mutational spectrum observed in the mutant strains and reveal that NucS-dependent MMR specific task is to eliminate G/T mismatches generated by the DNA polymerase during replication. Interestingly, our data unveil a crescent-shaped distribution of the transition frequency from the replication origin towards the chromosomal ends, shedding light on a possible link between NucS-mediated DSBs and Streptomyces genome evolution. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/572499v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1aa491dorg.highwire.dtl.DTLVardef@a62657org.highwire.dtl.DTLVardef@1f1d2aorg.highwire.dtl.DTLVardef@b3b389_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Structuring effects of archaeal replication origins

Archaea use eukaryotic-like DNA replication proteins to duplicate circular chromosomes similar to those of bacteria. Although archaeal replication origins have been maintained during the evolution, they are non-essential under laboratory conditions. Here we propose the local deviations from Chargaffs second parity rule of archaeal chromosomes result from the biased gene orientation and not from mutational biases. Our computational and experimental analyses indicate that the archaeal replication origins prevent head-to-head collisions of replication and transcription complexes as well as participate in coordination of the transfer of genetic information. Our results therefore suggest that the archaeal replication origins have alternative functions not related to their role in initiation of DNA replication.

microbiology↗

bioRxiv2020

CRISPR-mediated biocontainment

We have exploited the repetitive nature of transposable elements of the human genome to generate synthetic circuits. Transposable elements such as LINE-1 and Alu have successfully replicated in mammalian genomes throughout evolution to reach a copy number ranging from thousands to more than a million. Targeting these repetitive elements with programmable DNA nucleases such as CRISPR-Cas9 rapidly induce extremely high levels of cell death. We use this genotoxic feature to build synthetic biocontainment circuits: CRISPR defense system (CRISPR-DS) capable of preventing CRISPR genome editing, and we introduce the proof-of-concept of CRISPR Safety-Switch, an inducible, stringent and non-leaky kill-switch capable of clearing out cell lines resistant to DNA breaks.

synthetic biology↗

bioRxiv2019

Enabling large-scale genome editing by reducing DNA nicking

To extend the frontier of genome editing and enable the radical redesign of mammalian genomes, we developed a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks (DSBs) and single-strand breaks (SSBs). We used a set of gRNAs targeting repetitive elements - ranging in target copy number from about 31 to 124,000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ~13,200 and ~2610 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.\n\nOne Sentence SummaryBase editing with reduced DNA nicking allows for the simultaneous editing of >10,000 loci in human cells.

synthetic biology↗