A SRS2 homolog from Arabidopsis thaliana disrupts recombinogenic DNA intermediates and facilitates single strand annealing

Genetic and biochemical analyses of SRS2 homologs in fungi indicate a function in the processing of homologous recombination (HR) intermediates. To date, no SRS2 homologs have been described and analyzed in higher eukaryotes. Here, we report the first biochemical characterization of an SRS2 homolog from a multicellular eukaryote, the plant Arabidopsis thaliana. We studied the basic properties of AtSRS2 and were able to show that it is a functional 3′- to 5′-helicase. Furthermore, we characterized its biochemical function on recombinogenic intermediates and were able to show the unwinding of nicked Holliday junctions (HJs) and partial HJs (PX junctions). For the first time, we demonstrated strand annealing activity for an SRS2 homolog and characterized its strand pairing activity in detail. Our results indicate that AtSRS2 has properties that enable it to be involved in different steps during the processing of recombination intermediates. On the one hand, it could be involved in the unwinding of an elongating invading strand from a donor strand, while on the other hand, it could be involved in the annealing of the elongated strand at a later step

Probing Electric Fields in Protein Cavities by Using the Vibrational Stark Effect of Carbon Monoxide

To determine the magnitude and direction of the internal electric field in the Xe4 cavity of myoglobin mutant L29W-S108L, we have studied the vibrational Stark effect of carbon monoxide (CO) using infrared spectroscopy at cryogenic temperatures. CO was photodissociated from the heme iron and deposited selectively in Xe4. Its infrared spectrum exhibits Stark splitting into two bands associated with CO in opposite orientations. Two different photoproduct states can be distinguished, C′ and C″, with markedly different properties. For C′, characteristic temperature-dependent changes of the area, shift, and width were analyzed, based on a dynamic model in which the CO performs fast librations within a double-well model potential. For the barrier between the wells, a height of ∼1.8 kJ/mol was obtained, in which the CO performs oscillations at an angular frequency of ∼25 cm(−1). The magnitude of the electric field in the C′ conformation was determined as 11.1 MV/cm; it is tilted by an angle of 29° to the symmetry axis of the potential. Above 140 K, a protein relaxation leads to a significantly altered photoproduct, C″, with a smaller Stark splitting and a more confining potential (barrier >4 kJ/mol) governing the CO librations