Discoveries

Unique Properties of DNA Gyrase for (+) Supercoil Processing

October 2, 2017

Unique Properties of DNA Gyrase for (+) Supercoil Processing

The twisting of DNA in the same or opposite direction of the turn of the double helix gives rise to (+) or (-) supercoiling, respectively. Supercoiling exerts a strain on the helix, leading to the formation of loops, knots, and tangles that can have a major impact on the ability of DNA processing enzymes to carry out their function. This is especially true with regard to (+) supercoils that form ahead of the replication or transcription machinery as a result of DNA unwinding. In bacteria, (+) supercoils are removed through the action of two enzymes, gyrase and topoisomerase IV. Both enzymes work by grasping a strand of double-stranded DNA, introducing a break in that strand, and then passing a second strand through the break before it is reconnected. Topoisomerase IV can remove (-) as well as (+) supercoils, whereas gyrase introduces (-) supercoils in relaxed DNA. This difference in (-) supercoil processing is associated with the ability of gyrase, but not topoisomerase IV to wrap the second strand of DNA before passing it through the break. Relatively little is known, however, about how the two enzymes process (+) supercoils, leading Vanderbilt Basic Sciences investigator Neil Osheroff and his graduate student Rachel Ashley to take a closer look. They discovered that gyrase removes (+) supercoils much faster than it introduces (-) supercoils, and that it does so in bursts of activity as fast as 107 supercoils/s. A mutant enzyme unable to wrap DNA exhibited markedly reduced efficiency in (+) supercoil processing, indicating that wrapping was required for both of gyrase’s primary functions. Topoisomerase IV also removed (+) supercoils more quickly than (-) ones, but it was much less efficient than gyrase at (+) supercoil processing. When gyrase or topoisomerase IV break the first bound strand of DNA, it becomes covalently bound to the enzyme, forming a cleavage complex. Failure to rejoin the ends of the DNA strand can lead to double strand breaks and ultimately the death of the cell. Quinolone antimicrobial agents work by stabilizing the cleavage complex. The researchers showed that in the presence of quinolones, gyrase forms fewer double strand breaks while processing (+) supercoiled DNA than (-) supercoiled DNA. For topoisomerase IV, there was little difference in break formation between the two substrates. The results suggest that gyrase is particularly well-suited to process (+) supercoils formed ahead of the replication or transcription machinery in terms of both the rapidity of the process and the low rate of double strand break formation. The work is published in the journal Nucleic Acids Research [R. E. Ashley, et al. Nuc. Acids Res., 45, 9611].

A Super-Resolution Look at the Contractile Ring

September 22, 2017

During cell division, a ring of proteins forms around the equator of the cell. Then, after the chromosomes have been distributed to each pole of the mitotic spindle, the ring constricts, separating the cell into two daughter cells. This contractile ring is a complex structure that uses the mechanical energy of myosin motors to constrict a plasma membrane-associated network of F-actin fibers. Numerous studies have delineated the steps of contractile ring formation, starting with membrane-bound precursor nodes that eventually coalesce into a contiguous ring prior to constriction. However, a detailed understanding of contractile ring architecture is lacking, thereby preventing a complete understanding of its composition, structure and function. To fill this knowledge gap, Basic Sciences investigator Kathleen Gould and her laboratory utilized resources provided by Vanderbilt’s new Nikon Center of Excellence to obtain high resolution structural data on contractile ring structure in dividing Schizosaccharomyces pombe. The investigators individually expressed in yeast cells a total of 29 known contractile ring proteins linked at either their N- or C-terminus to a photoactivatable probe. The cells also expressed a distinct membrane-associated photoactivatable probe. This enabled the researchers to use fluorescence photoactivation localization microscopy (fPALM), a super-resolution microscopy technique to localize each protein in terms of its distance from the membrane. The results demonstrated three structurally distinct regions at 0-80 nm, 80-160 nm, and 160-400 nm from the membrane’s cytosolic surface. In the layer closest to the membrane were membrane-associated scaffold proteins and the tail of myosin II. The intermediate layer contained a complex network of accessory proteins and signaling molecules. The layer most distant from the membrane comprised predominantly F-actin along with the motor heads of myosin and an F-actin cross-linker. The researchers also found that some proteins, particularly those close to the membrane, formed clusters of various sizes, whereas others were more uniformly distributed, especially in the most distant layer. These findings provide a critical foundation for the better understanding of contractile ring assembly and function. The work is published in the journal eLife [N. A. McDonald, et al., (2017) eLife., 6, e28865].

Genetic Heterogeneity in Colorectal Cancer Metastases

September 22, 2017

Genetic Heterogeneity in Colorectal Cancer Metastases

Despite recent progress in early diagnosis and treatment, colorectal cancer remains a major cause of morbidity and mortality, with 90% of the deaths attributable to metastatic disease. We now know that cancer is largely a disease of genetic mutations and that most cancers harbor large numbers of different mutations. More recently, extensive whole genome sequencing has revealed that many tumors comprise multiple subclones of cells, each carrying a distinct set of genetic mutations – a property known as intratumor heterogeneity (ITH). ITH plays an important role in a cancer’s response to therapy and its ability to develop resistance to therapy, yet most studies of ITH have focused only on the primary tumor, yielding little information regarding the all-important metastases. This led Vanderbilt Basic Sciences investigator Bingshan Li, his collaborator Hushan Yang (Jefferson University, Philadelphia, PA) and their colleagues to conduct a detailed genomic analysis of 10 primary tumor, 3 positive lymph node, 10 metastatic tumor, and 5 normal tissue samples from 4 patients. Their results show that there is greater intertumor heterogeneity between patients than ITH within a given patient’s primary tumor. Furthermore, metastatic tumors demonstrate lower levels of ITH than their corresponding primary tumors. Of greater interest, however, was the finding that the metastatic tumors from a single patient are genetically distinct from each other and appear to have arisen from multiple clones within the primary tumor. Similarly, metastatic tumors appear to have distinct origins from cancer cells found within a positive lymph node from the same patient. These findings support the hypothesis that individual colorectal cancer metastases result from multiple separate seeding events, and that few arise from tumor cells present in lymph nodes. The relatively low level of ITH within metastases suggests that an individual sample might yield adequate genetic information to predict its response to therapy, but there is no guarantee that any individual sample will be representative of either the complete primary tumor or all metastatic foci. The work is published in the journal Annals of Oncology [Q. Wei, et al., (2017) Ann. Oncol., 28, 2138].

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