The C-Terminal Domain of the MutL Homolog from Neisseria gonorrhoeae Forms an Inverted Homodimer

The mismatch repair (MMR) pathway serves to maintain the integrity of the genome by removing mispaired bases from the newly synthesized strand. In E. coli, MutS, MutL and MutH coordinate to discriminate the daughter strand through a mechanism involving lack of methylation on the new strand. This facilitates the creation of a nick by MutH in the daughter strand to initiate mismatch repair. Many bacteria and eukaryotes, including humans, do not possess a homolog of MutH. Although the exact strategy for strand discrimination in these organisms is yet to be ascertained, the required nicking endonuclease activity is resident in the C-terminal domain of MutL. This activity is dependent on the integrity of a conserved metal binding motif. Unlike their eukaryotic counterparts, MutL in bacteria like Neisseria exist in the form of a homodimer. Even though this homodimer would possess two active sites, it still acts a nicking endonuclease. Here, we present the crystal structure of the C-terminal domain (CTD) of the MutL homolog of Neisseria gonorrhoeae (NgoL) determined to a resolution of 2.4 Å. The structure shows that the metal binding motif exists in a helical configuration and that four of the six conserved motifs in the MutL family, including the metal binding site, localize together to form a composite active site. NgoL-CTD exists in the form of an elongated inverted homodimer stabilized by a hydrophobic interface rich in leucines. The inverted arrangement places the two composite active sites in each subunit on opposite lateral sides of the homodimer. Such an arrangement raises the possibility that one of the active sites is occluded due to interaction of NgoL with other protein factors involved in MMR. The presentation of only one active site to substrate DNA will ensure that nicking of only one strand occurs to prevent inadvertent and deleterious double stranded cleavage

Prediction and Testing of Biological Networks Underlying Intestinal Cancer

Colorectal cancer progresses through an accumulation of somatic mutations, some of which reside in so-called “driver” genes that provide a growth advantage to the tumor. To identify points of intersection between driver gene pathways, we implemented a network analysis framework using protein interactions to predict likely connections – both precedented and novel – between key driver genes in cancer. We applied the framework to find significant connections between two genes, Apc and Cdkn1a (p21), known to be synergistic in tumorigenesis in mouse models. We then assessed the functional coherence of the resulting Apc-Cdkn1a network by engineering in vivo single node perturbations of the network: mouse models mutated individually at Apc (Apc1638N+/−) or Cdkn1a (Cdkn1a−/−), followed by measurements of protein and gene expression changes in intestinal epithelial tissue. We hypothesized that if the predicted network is biologically coherent (functional), then the predicted nodes should associate more specifically with dysregulated genes and proteins than stochastically selected genes and proteins. The predicted Apc-Cdkn1a network was significantly perturbed at the mRNA-level by both single gene knockouts, and the predictions were also strongly supported based on physical proximity and mRNA coexpression of proteomic targets. These results support the functional coherence of the proposed Apc-Cdkn1a network and also demonstrate how network-based predictions can be statistically tested using high-throughput biological data

The saltation illusion demonstrates integrative processing of spatiotemporal information in thermoceptive and nociceptive networks

In sensory saltation, first reported by Geldard and Sherrick (Science 178:178-179, 1972), a stimulus is displaced towards a second one following closely in time and space as a function of the delay between the stimuli. The distance between stimulus locations is restricted by the extension of sensory fields in the primary somatosensory cortex. Saltation is assumed to reflect dynamic changes in these cortical representations. The present study demonstrates for the first time saltation in thermoceptive and nociceptive pathways with CO(2) laser stimulation. Stimuli were presented to the dorsal forearms of 18 healthy subjects at two intensities. Saltation patterns consisted of a reference stimulus S0 near the wrist, the first test stimulus S1 at the reference location after a fixed onset delay of 1,000 ms, and a second test stimulus S2 at a location 105 mm distant from reference after a variable onset delay of 60-516 ms. Perceived positions were indicated by the subjects without skin contact with a 3D tracker. As expected, subjects mislocalized S1 towards S2. Mean S1 displacement was 51+/-36 mm. Decreasing delays between S1 and S2 resulted in increasing displacements, independent of intensity. However, since no clear-cut discrimination of thermal versus nociceptive activation could be achieved definite conclusions about differences between the two modalities cannot be drawn. In addition, effects of body site on the saltation characteristics were observed. The saltation paradigm constitutes a promising approach to the functional analysis of spatiotemporal dynamics in thermoceptive and nociceptive networks to supplement brain-mapping approaches to cortical sensory fields