Biology subjects

Heiter, D.

Publications and source records attributed to Heiter, D..

3 recordsLinked to original sources

bioRxiv2023

FFPE DNA shows two major error profiles derived from deamination of cytosine and methylcytosine that can be mitigated using distinct repair strategies.

Avoiding damage-induced sequencing errors is a critical step for the accurate identification of medium to rare frequency mutations in DNA samples. In the case of FFPE samples, deamination of cytosine moieties represents a major damage resulting in the loss of DNA material and sequencing errors. In this study, we demonstrated that, while damage from deamination of both cytosine and methylated cytosine moieties results in elevated C to T transition, the error profiles and mediation strategies are different and easily distinguishable. While damage-induced sequencing errors from cytosine deamination is driven by the end-repair step commonly used in NGS workflow, DNA damage resulting from deamination of methylated cytosine is another major contributor to sequencing errors at CpG sites. Uracil DNA glycosylase and human thymine DNA glycosylase can respectively eliminate and mitigate both damages in FFPE DNA samples, therefore increasing sequencing accuracy notably for the identification of moderate allelic frequency variants.

genomics↗

bioRxiv2022

Modification-Dependent Restriction Endonuclease-based sequencing method (EcoWI-seq) maps the genome-wide landscape of phosphorothioate modification at base resolution.

Phosphorothioation (PT), in which a non-bridging oxygen is replaced by a sulfur, is one of the rare modifications discovered in bacteria and archaea that occurs on the sugar-phosphate backbone as opposed to the nucleobase moiety of DNA. While PT modification is widespread in the prokaryotic kingdom, how PT modifications are distributed in the genomes and their exact roles in the cell remain to be defined. In this study, we developed a simple and convenient technique called EcoWI-seq based on a modification-dependent restriction endonuclease to identify genomic positions of PT modifications. EcoWI-seq shows similar performance than other PT modification detection techniques and additionally, is easily scalable while requiring little starting material. As a proof of principle, we applied EcoWI-seq to map at base resolution the PT modifications in the genomes of both the Salmonella enterica cerro 87 and E. coli expressing the dnd+ gene cluster. Specifically, we address whether the partial establishment of modified PT positions is a stochastic or deterministic process. EcoWI-seq reveals a systematic usage of the same subset of target sites in clones for which the PT modification has been independently established.

microbiology↗

bioRxiv2022

Expression and Purification of BsaXI Restriction Endonuclease and Engineering New Specificity from BsaXI Specificity (S) Subunit

BsaXI is a Type IIB restriction endonuclease (REase) that cleaves both sides of its recognition sequence 5 {downarrow}N9 AC N5 CTCC N10{downarrow} 3 (complement strand 5 {downarrow}N7 GGAG N5 GT N12{downarrow} 3), creating 3-base 3 overhangs. Here we report the cloning and expression of bsaXIS and bsaXIRM genes in E. coli. BsaXI activity was successfully reconstituted by mixing the BsaXI RM fusion subunit with the BsaXI S subunit and the enzyme complex further purified by chromatography over 6 columns. As expected, the S subunit consisted of two subdomains encoding TRD1-CR1 (TRD, target recognition domain, CR, conserved region) for 5 AC 3, and TRD2-CR2 presumably specifying 5 CTCC 3. TRD1-CR1 (TRD2-CR2 deletion) or duplication of TRD1 (TRD1-CR1-TRD1-CR2) both generated a new specificity 5 AC N5 GT 3 when the S variants were complexed with the RM subunits. Circular permutation of TRD1 and TRD2, i.e. relocation of TRD2-CR2 to the N-terminus and TRD1-CR1 to the C-terminus generated the same specificity with the RM subunits, although some wobble cleavage was detected. The TRD2 domain in the BsaXI S subunit can be substituted by a close homolog ([~]59% sequence identity) and generated the same specificity. However, TRD2-CR2 domain alone failed to express in E. coli, but CR1-TRD2-CR2 protein could be expressed and purified which showed partial nicking activity with the RM subunits. This work demonstrated that like Type I restriction systems, the S subunit of a Type IIB system could also be manipulated to create new specificities. Genome mining of BsaXI TRD2 homologs in GenBank found more than 36 orphan TRD2 homologs, implying that quite a few orphan TRD2s are present in microbial genomes that may be potentially paired with other TRDs to create new restriction specificities.

microbiology↗