Protein-DNA complexes can impede DNA replication and cause replication fork collapse. Whilst it is known that homologous recombination is deployed in such instances to restart replication, it is unclear how a stalled fork transitions into a collapsed fork at which recombination proteins can load. Previously we established assays in Schizosaccharomyces pombe for studying recombination induced by replication fork collapse at the site-specific protein-DNA barrier RTS1 (Nguyen et al., 2015). Here, we provide evidence that efficient recruitment/retention of two key recombination proteins (Rad51 and Rad52) to RTS1 depends on unloading of the polymerase sliding clamp PCNA from DNA by Elg1. We also show that, in the absence of Elg1, reduced recombination is partially suppressed by deleting fbh1 or, to a lesser extent, srs2, which encode known anti-recombinogenic DNA helicases. These findings suggest that PCNA unloading by Elg1 is necessary to limit Fbh1 and Srs2 activity, and thereby enable recombination to proceed.

L-myo-inositol-1-phosphate synthase (MIPS; EC: 5.5.1.4) catalyzes the conversion of D-glucose-6-phosphate to 1L-myo-inositol-1-phosphate, the rate limiting step in the biosynthesis of all inositol containing compounds. Myo-inositol and its derivatives are implicated in membrane biogenesis, cell signaling, salinity stress tolerance and a number of other metabolic reactions in different organisms. This enzyme has been reported from a number of bacteria, fungi, plants and animals. In the present study some bryophytes available in the Eastern Himalaya have been screened for free myo-inositol content. It is seen that Bryum coronatum, a bryopsid shows the highest content of free myo-inositol among the species screened. Subsequently , the enzyme MIPS has been partially purified to the tune of about 70 fold with approximately 18% recovery form the reproductive part bearing gametophytes of Lunularia cruciata. The L. cruciata synthase specifically utilized D-glucose-6-phosphate and NAD + as its substrate and co-factor respectively. The optimum pH shown was 7.0 while the temperature maximum was at 30˚C.

It is thought that many of the simple and complex genomic rearrangements associated with congenital diseases and cancers stem from mistakes made during the restart of collapsed replication forks by recombination enzymes. It is hypothesised that this recombination-mediated restart process transitions from a relatively accurate initiation phase to a less accurate elongation phase characterised by extensive template switching between homologous, homeologous and microhomologous DNA sequences. Using an experimental system in fission yeast, where fork collapse is triggered by a site-specific replication barrier, we show that ectopic recombination, associated with the initiation of recombination-dependent replication (RDR), is driven mainly by the Rad51 recombinase, whereas template switching, during the elongation phase of RDR, relies more on DNA annealing by Rad52. This finding provides both evidence and a mechanistic basis for the transition hypothesis.

Consequently, the following was added to the Author Contributions: A.K., S.T., M.O.N., J.O., and M.J.: conceived and performed experiments. E.B. and C.A. performed experiments; C.A.M. and F.O. provided reagents, expertise, and feedback; M.C.W. conceived experiments, wrote the paper, and secured funding'. This has been corrected in both the PDF and HTML versions of the Article.

No abstract