Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent \u3cem\u3ePlasmodium falciparum\u3c/em\u3e SSB as a Reporter for Single-Stranded DNA

Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism

Role of a Conserved Glutamate Residue in the \u3cem\u3eEscherichia coli\u3c/em\u3e SecA ATPase Mechanism

Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the “DEVD” Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018–2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s–1versus 27 s–1, respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439–17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis

AN INTRODUCTION TO PROBABILITY DISTRIBUTIONS

Probability allows us to infer from a sample to a population. In fact, inference is a tool of probability theory. This paper looks briefly at the Binomial, Poisson, and Normal distributions. These are probability distributions, which are used extensively in inference. An understanding of these distributions will assist the chiropractor and osteopath to critically appraise the literature

THE t TEST: An Introduction

The t distribution is a probability distribution similar to the Normal distribution. It is commonly used to test hypotheses involving numerical data. This paper provides an understanding of the t distribution and uses a musculo-skeletal example to illustrate its application

CMB lensing and primordial squeezed non-Gaussianity

Squeezed primordial non-Gaussianity can strongly constrain early-universe physics, but it can only be observed on the CMB after it has been gravitationally lensed. We give a new simple non-perturbative prescription for accurately calculating the effect of lensing on any squeezed primordial bispectrum shape, and test it with simulations. We give the generalization to polarization bispectra, and discuss the effect of lensing on the trispectrum. We explain why neglecting the lensing smoothing effect does not significantly bias estimators of local primordial non-Gaussianity, even though the change in shape can be >~10%. We also show how tau_NL trispectrum estimators can be well approximated by much simpler CMB temperature modulation estimators, and hence that there is potentially a ~10-30% bias due to very large-scale lensing modes, depending on the range of modulation scales included. Including dipole sky modulations can halve the tau_NL error bar if kinematic effects can be subtracted using known properties of the CMB temperature dipole. Lensing effects on the g_NL trispectrum are small compared to the error bar. In appendices we give the general result for lensing of any primordial bispectrum, and show how any full-sky squeezed bispectrum can be decomposed into orthogonal modes of distinct angular dependence.Comment: 22 pages, 6 figures; minor edits to match published versio

Millennial‐Scale Vulnerability of the Antarctic Ice Sheet to Regional Ice Shelf Collapse

The response of the Antarctic Ice Sheet to ice shelf collapse is explored with a high resolution ice sheet model. Rapid melting is applied to each of its major present day drainage basins in turn , to determine which parts of the ice sheet are most vulnerable to change in oceanic forcing, over the next 1000 years. We findthat West Antarctica can be largely deglaciated over a millenium, leading to more than two metres of sea level rise, if any of its major ice shelved disintegrated. The response of East Antarctica is more muted, but not negligible