RESUMO
SAW1 is required by the Rad1-Rad10 nuclease for efficient removal of 3' non-homologous DNA ends (flaps) formed as intermediates during two modes of double-strand break repair in S. cerevisiae, single-strand annealing (SSA) and synthesis-dependent strand annealing (SDSA). Saw1 was shown in vitro to exhibit increasing affinity for flap DNAs as flap lengths varied from 0 to 40 deoxynucleotides (nt) with almost no binding observed when flaps were shorter than 10 nt. Accordingly, our prior in vivo fluorescence microscopy investigation showed that SAW1 was not required for recruitment of Rad10-YFP to DNA double-strand breaks (DSBs) when flaps were â¼10 nt, but it was required when flaps were â¼500 nt in G1 phase of the cell cycle. We were curious whether we would also observe an increased requirement of SAW1 for Rad10 recruitment in vivo as flaps varied from â¼20 to 50 nt, as was shown in vitro. In this investigation, we utilized SSA substrates that generate 20, 30, and 50 nt flaps in vivo in fluorescence microscopy assays and determined that SAW1 becomes increasingly necessary for SSA starting at about â¼20 nt and is completely required at â¼50 nt. Quantitative PCR experiments corroborate these results by demonstrating that repair product formation decreases in the absence of SAW1 as flap length increases. Experiments with strains containing fluorescently labeled Saw1 (Saw1-CFP) show that Saw1 localizes with Rad10 at SSA foci and that about half of the foci containing Rad10 at DSBs do not contain Saw1. Colocalization patterns of Saw1-CFP are consistent regardless of the flap length of the substrate and are roughly similar in all phases of the cell cycle. Together, these data show that Saw1 becomes increasingly important for Rad1-Rad10 recruitment and SSA repair in the â¼20-50 nt flap range, and Saw1 is present at repair sites even when not required and may depart the repair site ahead of Rad1-Rad10.
RESUMO
Prion protein, PrP(C), is a glycoprotein that is expressed on the cell surface beginning with the early stages of embryonic stem cell differentiation. Previously, we showed that ectopic expression of PrP(C) in human embryonic stem cells (hESCs) triggered differentiation toward endodermal, mesodermal, and ectodermal lineages, whereas silencing of PrP(C) suppressed differentiation toward ectodermal but not endodermal or mesodermal lineages. Considering that PrP(C) might be involved in controlling the balance between cells of different lineages, the current study was designed to test whether PrP(C) controls differentiation of hESCs into cells of neuron-, oligodendrocyte-, and astrocyte-committed lineages. PrP(C) was silenced in hESCs cultured under three sets of conditions that were previously shown to induce hESCs differentiation into predominantly neuron-, oligodendrocyte-, and astrocyte-committed lineages. We found that silencing of PrP(C) suppressed differentiation toward all three lineages. Similar results were observed in all three protocols, arguing that the effect of PrP(C) was independent of differentiation conditions employed. Moreover, switching PrP(C) expression during a differentiation time course revealed that silencing PrP(C) expression during the very initial stage that corresponds to embryonic bodies has a more significant impact than silencing at later stages of differentiation. The current work illustrates that PrP(C) controls differentiation of hESCs toward neuron-, oligodendrocyte-, and astrocyte-committed lineages and is likely involved at the stage of uncommitted neural progenitor cells rather than lineage-committed neural progenitors.