Lauren Higgins

Cofactor requirement of HpyAV restriction endonuclease.

Background Helicobacter pylori is the etiologic agent of common gastritis and a risk factor for gastric cancer. It is also one of the richest sources of Type II restriction-modification (R-M) systems in microorganisms. Principal Findings We have cloned, expressed and purified a new restriction endonuclease HpyAV from H. pylori strain 26695. We determined the HpyAV DNA recognition sequence and cleavage site as CCTTC 6/5. In addition, we found that HpyAV has a unique metal ion requirement: its cleavage activity is higher with transition metal ions than in Mg++. The special metal ion requirement of HpyAV can be attributed to the presence of a HNH catalytic site similar to ColE9 nuclease instead of the canonical PD-X-D/EXK catalytic site found in many other REases. Site-directed mutagenesis was carried out to verify the catalytic residues of HpyAV. Mutation of the conserved metal-binding Asn311 and His320 to alanine eliminated cleavage activity. HpyAV variant H295A displayed approximately 1% of wt activity. Conclusions/Significance Some HNH-type endonucleases have unique metal ion cofactor requirement for optimal activities. Homology modeling and site-directed mutagenesis confirmed that HpyAV is a member of the HNH nuclease family. The identification of catalytic residues in HpyAV paved the way for further engineering of the metal binding site. A survey of sequenced microbial genomes uncovered 10 putative R-M systems that show high sequence similarity to the HpyAV system, suggesting lateral transfer of a prototypic HpyAV-like R-M system among these microorganisms.

Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants

BmrI (ACTGGG N5/N4) is one of the few metal-independent restriction endonucleases (REases) found in bacteria. The BmrI restriction-modification system was cloned by the methylase selection method, inverse PCR, and PCR. BmrI REase shows significant amino acid sequence identity to BfiI and a putative endonuclease MspBNCORF3798 from the sequenced Mesorhizobium sp. BNC1 genome. The EDTA-resistant BmrI REase was successfully over-expressed in a pre-modified E. coli strain from pET21a or pBAC-expIQ vectors. The recombinant BmrI REase shows strong promiscuous activity (star activity) in NEB buffers 1, 4, and an EDTA buffer. Star activity was diminished in buffers with 100-150 mM NaCl and 10 mM MgCl(2). His-tagged BmrI192, the N-terminal cleavage domain of BmrI, was expressed in E. coli and purified from inclusion bodies. The refolded BmrI192 protein possesses non-specific endonuclease activity. BmrI192 variants with a single Ser to Cys substitution (S76C or S90C) and BmrI200 (T200C) with a single Cys at the C-terminal end were also constructed and purified. BmrI200 digests both single-strand (ss) and double-strand (ds) DNA and the nuclease activity on ss DNA is at least 5-fold higher than that on ds DNA. The Cys-containing BmrI192 and BmrI200 nuclease variants may be useful for coupling to other DNA binding elements such as synthetic zinc fingers, thio-containing locked nucleic acids (LNA) or peptide nucleic acids (PNA).

Abstract 1425: A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples

Next-generation sequencing (NGS) methods are used extensively to profile mutations present in diseased human tissues. These genomic approaches hold great promise for personalized medicine but sequencing accuracy is essential for proper patient diagnosis and determining a treatment plan. A common source of DNA for genomic profiling is formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from patient biopsy. FFPE DNA poses important challenges for preparing NGS libraries including low input amounts and poor DNA quality, resulting from extensive fixation- and storage-induced DNA damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-causing variants from noise. We previously showed that a major fraction of somatic mutations described in publicly available datasets are due to such sequencing artifacts (Chen et al., Science 2017). Furthermore, we showed that enzymatic repair of DNA before library preparation improves the library quality and reduces background noise. We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries. Additionally, we tested the efficacy of the V2 repair mix in improving sequencing accuracy for FFPE DNA samples obtained from different cancer tissues. We performed target enrichment on a panel of 151 cancer genes, deep sequenced, and performed variant analysis. For a subset of variants, we further validated our results using a droplet digital PCR (ddPCR) assay. Both methods showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls. Therefore, enzymatic repair is a critical first step in preparing FFPE DNA sequencing libraries, allowing more sensitive and robust detection of low frequency, disease variants. Citation Format: Pingfang Liu, Margaret Heider, Chen Song, Lixin Chen, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1425.