Browsing by Subject "mutagenesis"

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Directed Evolution Of Xylose Isomerase For Improved Xylose Catabolism And Fermentation In The Yeast Saccharomyces Cerevisiae
(2012-08) Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S.; Lee, Sun-Mi; Jellison, Taylor; Alper, Hal S.
The heterologous expression of a highly functional xylose isomerase pathway in Saccharomyces cerevisiae would have significant advantages for ethanol yield, since the pathway bypasses cofactor requirements found in the traditionally used oxidoreductase pathways. However, nearly all reported xylose isomerase-based pathways in S. cerevisiae suffer from poor ethanol productivity, low xylose consumption rates, and poor cell growth compared with an oxidoreductase pathway and, additionally, often require adaptive strain evolution. Here, we report on the directed evolution of the Piromyces sp. xylose isomerase (encoded by xylA) for use in yeast. After three rounds of mutagenesis and growth-based screening, we isolated a variant containing six mutations (E15D, E114G, E129D, T142S, A177T, and V433I) that exhibited a 77% increase in enzymatic activity. When expressed in a minimally engineered yeast host containing a gre3 knockout and tall and XKS1 overexpression, the strain expressing this mutant enzyme improved its aerobic growth rate by 61-fold and both ethanol production and xylose consumption rates by nearly 8-fold. Moreover, the mutant enzyme enabled ethanol production by these yeasts under oxygen-limited fermentation conditions, unlike the wild-type enzyme. Under microaerobic conditions, the ethanol production rates of the strain expressing the mutant xylose isomerase were considerably higher than previously reported values for yeast harboring a xylose isomerase pathway and were also comparable to those of the strains harboring an oxidoreductase pathway. Consequently, this study shows the potential to evolve a xylose isomerase pathway for more efficient xylose utilization.
Introducing the non-B DNA Motif Search Tool (nBMST)
(Genome Biology, 2011-09-19) Cer, Regina Z; Bruce, Kevin H.; Donohue, Duncan E.; Temiz, Alpay N.; Bacolla, Albino; Mundunuri, Uma S.; Yi, Ming; Volfovsky, Natalia; Luke, Brian T.; Collins, Jack R.; Stephens, Robert M.
DNA sequence motifs with the ability to form non-B (non-canonical) structures have been linked to a variety of regulatory and pathological processes. Although the exact mechanism is unknown, recent work has provided significant evidence that non-B DNA structures may play a role in DNA instability and mutagenesis, leading to both DNA rearrangements and increased mutational rates, which are hallmarks of cancer. We have developed algorithms to identify a wide variety of non-B-DNA-forming motifs, including G-quadruplex-forming repeats, direct repeats and slipped motifs, inverted repeats and cruciform motifs, mirror repeats and triplex motifs, and A-phased repeats. After identifying these motifs in the mammalian reference genomes of human, mouse, chimpanzee, macaque, cow, dog, rat and platypus, the data were made publicly available in non-B DB [1]. However, it soon became apparent that it was not feasible to annotate the ever-growing list of genomic data and that it would be more effective to provide researchers with a systematic tool to predict these motifs in their own genomic data. Thus, the non-B DNA Motif Search Tool (nBMST) was created, and it is freely available online [2]. nBMST is a web interface that enables researchers to interactively submit any DNA sequence for searching for non-B DNA motifs. Once a user submits one or more DNA sequences in FASTA format, nBMST returns a comprehensive results page that contains the following: downloadable files in both a tab-delimited format and a generic feature format (GFF); a visualization, including PNG images; and a dynamic genome browser created using the Generic Genome Browser (GBrowse) [3] (version 2.0). Currently, nBMST allows file sizes of up to 20 MB of DNA sequence to be uploaded and stores the results for registered users for up to six months. In summary, the purpose of nBMST is to help provide insight into the involvement of alternative DNA conformations in cancer and other diseases, as well as into other potential biological functions.
Short inverted repeats are hot spots for genetic instability in mammalian cells
(2012) Lu, Steve; Vasquez, Karen M.; Laude, David
Analyses of chromosomal aberrations in genetics disorders such as leukemia and lymphoma have provided compelling evidence that segments of the human genome containing repetitive sequences can be prone to breakage and chromosomal rearrangements. While long inverted repeat (IR) sequences have been shown to be mutagenic in mammalian cells, the mutagenic potential of short IR sequences (<30 bp), which are very abundant in eukaryotic genomes and often co-localize near chromosomal breakpoints, has not been investigated. Herein, we demonstrate that cruciform structures formed at short perfect IRs are mutagenic in mammalian cells. We found that DNA double-strand breaks (DSBs) occurred in vivo at or near the IR sequences, and the majority of the mutations induced by short IRs were large-scale deletions spanning the palindromic sequence. Analyses of the mutant junctions revealed that ~80% contained microhomologies, which are characteristic of an error-prone microhomology-mediated end-joining repair mechanism. We found that cruciform-induced mutations and DNA strand breaks may occur through 1) a replication-dependent mechanism involving DNA replication fork stalling or 2) replication-independent, structure-specific enzymes that facilitate cleavage at positions adjacent to the IR sequences. Taken together, our results demonstrate that short inverted repeat sequences play a role in genetic instability, and provide a plausible mechanistic explanation for the co-localization of IRs with chromosome breakage points in human disease. These findings also support the hypothesis that alternatively structured DNA is a feature of genome plasticity and may be a contributor to human disease and a driving force in the evolution of the human genome by providing a means for diversity within the population.