No abstract

The RecG DNA helicase a key player in stalled replication fork rescue. The single-stranded DNA binding protein (SSB) participates in this process, but its role in the interaction of RecG with the fork remains unclear. We used atomic force microscopy (AFM) to visualize the interaction of RecG with a fork DNA in the presence of SSB. We discovered that SSB enhances RecG loading efficiency onto the DNA fork by threefold. Additionally, SSB interacts with RecG leading to the RecG remodeling. As a result, RecG separates from the fork, but remains bound to the DNA duplex. Moreover, in this new binding mode RecG is capable of translocation along the parental duplex DNA. We propose a model of RecG interaction with the replication fork involving two RecG binding modes. SSB plays the role of a remodeling factor defining the mode of RecG binding to the fork mediated by the SSB C-terminus. In the translocating mode, RecG remains in the vicinity of the fork and is capable of initiating the fork regression. Our results afford novel mechanistic insights into RecG interaction with the replication fork and provide the basis for further structural studies.

It is now well established that mammalian heme proteins are reactive with various nitrogen oxide species and that these reactions may play significant roles in mammalian physiology. For example the ferrous heme protein myoglobin (Mb) has been shown to reduce nitrite (NO2−) to nitric oxide (NO) under hypoxic conditions. We demonstrate here that the distal pocket histidine residue (His64) of horse heart metMbIII (i.e., ferric MbIII) has marked effects on the mode of nitrite ion coordination to the iron center. X-ray crystal structures were determined for the mutant proteins metMbIII H64V (2.0 Å resolution) and its nitrite ion adduct metMbIII H64V-nitrite (1.95 Å resolution), and metMbIII H64V/V67R (1.9 Å resolution) and its nitrite ion adduct metMbIII H64V/V67R-nitrite (2.0 Å resolution). These are compared to the known structures of wild type hh metMbIII and its nitrite ion adduct hh metMbIII-nitrite, which binds NO2− via an O-atom in a trans-FeONO configuration. Unlike wt metMbIII, no axial H2O is evident in either of the metMbIII mutant structures. In the ferric H64V-nitrite structure, replacement of the distal His residue with Val alters the binding mode of nitrite from the nitrito (O-binding) form in the wild-type protein to a weakly bound nitro (N-binding) form. Reintroducing a H-bonding residue in the H64V/V67R double mutant restores the O-binding mode of nitrite. We have also examined the effects of these mutations on reactivities of the metMbIIIs with cysteine as a reducing agent and of the (ferrous) MbIIs with nitrite ion under anaerobic conditions. The MbIIs were generated by reduction of the MbIII precursors in a second order reaction with cysteine, the rate constants for this step following the order H64V/V67R > H64V >> wt. The rate constants for the oxidation of the MbIIs by nitrite (giving NO as the other product) follow the order wt > H64V/V67R >> H64V and suggest a significant role of the distal pocket H-bonding residue in nitrite reduction.

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 M ammonium sulfate must be present, bringing into question the validity of these data as these are non-physiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using purification of tagged complexes, our results demonstrate that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the “storage form” of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB which, by virtue of its high affinity for single stranded DNA, allows these helicases to out compete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.