Multiprotein E. coli SSB–ssDNA complex shows both stable binding and rapid dissociation due to interprotein interactions

Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.     

Time- and state-dependent effects of methanethiosulfonate ethylammonium (MTSEA) exposure differ between heart and skeletal muscle voltage-gated Na+ channels

The substituted-cysteine scanning method (SCAM) is used to study conformational changes in proteins. Experiments using SCAM involve site-directed mutagenesis to replace native amino acids with cysteine and subsequent exposure to a methanethiosulfonate (MTS) reagent such as methanethiosulfonate ethylammonium (MTSEA). These reagents react with substituted-cysteines and can provide functional information about relative positions of amino acids within a protein. In the human heart voltage-gated Na⁺ channel hNav1.5 there is a native cysteine at position C373 that reacts rapidly with MTS reagents resulting in a large reduction in whole-cell Na⁺ current (I_Na). Therefore, in order to use SCAM in studies in this isoform, this native cysteine is mutated to a non-reactive residue, e.g., tyrosine. This mutant, hNav1.5-C373Y, is resistant to the MTS-mediated decrease in I_Na. Here we show that this resistance is time- and state-dependent. With relatively short exposure times to MTSEA (<4 min), there is little effect on I_Na. However, with longer exposures (4–8 min), there is a large decrease in I_Na, but this effect is only found when hNav1.5-C373Y is inactivated (fast or slow) — MTSEA has little effect in the closed state. Additionally, this long-term, state-dependent effect is not seen in human skeletal muscle Na⁺ channel isoform hNav1.4, which has a native tyrosine at the homologous site C407. We conclude that differences in molecular determinants of inactivation between hNav1.4 and hNav1.5 underlie the difference in response to MTSEA exposure.     

Menage á trois: Double strand break repair,V(D)J recombination and DNA-PK

All organisms possess mechanisms to repair double strand breaks (dsbs) generated in their DNA by damaging agents. Site-specific dsbs are also introduced during V(D)J recombination. Four complementation groups of radiosensitive rodent mutants are defective in the repair of dsbs, and are unable to carry out V(D)J recombination effectively. The immune defect in Severe Combined Immunodeficient (scid) mice also results from an inability to undergo effective V(D)J recombination, and scid cell lines display a repair defect and belong to one of these complementation groups. These findings indicate a mechanistic overlap between the processes of DNA repair and V(D)J recombination. Recently, two of the genes defined by these complementation groups have been identified and shown to encode components of DNA-dependent protein kinase (DNA-PK). We review here the three fields which have become linked by these findings, and discuss the involvement of DNA-PK in dsb rejoining and in V(D)J recombination.     

Sampling error in non-invasive genetic analyses of an endangered social carnivore

Modern non-invasive genetic technologies are useful in studies of rare and difficult-to-observe species. An examination of endangered African wild dog (Lycaon pictus) faecal DNA revealed that 11.4% of samples were assigned incorrectly to an individual. Sampling mistakes in the field are not normally considered in non-invasive genetic assessments, but can be a significant source of error. To ensure meticulous data interpretation, non-invasive genetic studies should track and report sampling inaccuracies.     

Struggling with Development: The Politics of Hunger and Gender in the Philippines and Hunger and Shame: Child Malnutrition and Poverty on Mount Kilimanjaro

Struggling with Development: The Politics of Hunger and Gender in the Philippines. Lynn M. Kwiatkowski. Boulder, CO: Westview, 1998. xiv. 350 pp.
Hunger and Shame: Child Malnutrition and Poverty on Mount Kilimanjaro. Mary Howard and Ann V. Millard. New York: Routledge, 1997. xxiv. 271 pp.     

Active chloride transport in the leaf epidermis of Commelina communis in relation to stomatal activity

Summary A chloride selective micro-electrode has been used to determine vacuolar chloride concentrations in individual cells of the leaf epidermis of Commelina communis. When the stomata were open a gradient in chloride concentration across the stomatal complex was observed with the highest concentration in the guard cells. On stomatal closure the chloride gradient was reversed. Calculation of the driving forces on chloride indicated that active transport of chloride was occurring during both stomatal opening and closure. This transport appeared to be energetically independent of the transport of potassium. These results are discussed in relation to the behaviour of other anions during stomatal movements.