To continue, the 2D-gel membranes were hybridized with a second probe (Probe B), corresponding to the 35S rRNA gene (Figure 16A, left). Because the 35S rRNA gene does not contain an origin of replication, it must be replicated by RFs that enter the fragment from either direction, leading to Y-shaped molecules (Figure 16A, right). For the WT, the Y arc signal increases following release from the G1 block, peaking at 45 minutes and then the signal diminishes as replication is completed. In contrast, persistence of the Y arc and X spike signals was apparent throughout the time-course in the rnh1∆ rnh2∆ mutants. A particular accumulation of Y-structures towards the end of the descending Y-arc (to the right of the inflection point) was evident for the double mutant, and confirmed by quantification of right versus leftwards Y-arc signal (Figure 16B). This result is consistent with a slowdown of RFs through this highly transcribed gene region and would suggest that RF progression through the 35S rRNA gene was affected in the RNH-mutant.
Surprisingly, for the rnh1∆ rnh2∆ double mutant we noted the appearance of bubble-shaped molecules, most clearly seen at 105 minutes (indicated by blue arrow, Figure 16A; quantified in Figure 16B), but arising 75 minutes after release from CPT treatment.
Figure 16. CPT treatment of rnh1Δ rnh2Δ mutant cells provokes rARS-independent
replication initiation at late S-phase. A. Predicted (gray) and novel (black) replication
intermediates (right) following hybridization to the transcribed 35S gene (probe B). 2D-gels as for
Figure 15. but hybridized with probe B (left). Blue arrows represent bubble arcs; red arrows
represent sub-Y arcs. B. Quantification of RIs for CPT treated samples. Quantifications are relative
to the “n” spot intensity.
A bubble arc is indicative of active replication from an origin within this fragment, since only active replication within a fragment leads to the formation of replication bubbles in 2D-gel. Additional signals resembling Y-arcs were observed in the 2D-gels of the RNH double mutants, which were not observed in the WT and were dependent upon CPT treatment (Figure 17). These signals, apparent from 45 minutes, have been denominated sub-Y arcs (150), as they migrate below the standard Y-arc, (indicated by red arrows, Figure 16A).
Figure 17. rARS-independent replication initiation is only observed in RNH- mutants
following CPT treatment. 2D-gels as for Figure 6.
We wished to characterize these interesting 2D-gel results further. Firstly, in order to classify the pausing sites more precisely they were compared to the pause sites of a fob1∆ rrm3∆ mutant (Figure 18A). Work by Virginia Zakian´s group has described known pausing sites for the rrm3∆ mutant (147), a helicase needed to allow RF passage through protein-DNA interactions. We chose to use a fob1∆ rrm3∆ double mutant, to additionally remove the strong pause signal caused by the RFB, as RF blocking activity at the RFB is dependent upon the activity of Fob1. The majority of the pausing sites identified in the RNH- mutant in response to
CPT treatment overlapped with those previously described for DNA helicase-deficient rrm3∆ mutants (147). Consequently, the RFPs, (labelled a to e, Figure 18A), correspond to sites of protein barriers provided by the ribosomal ARS (sites a/b), or the RNA PolIII transcribed 5S gene (site c) or the 3’ end of RNA PolI transcribed 35S gene (site d). Notably one RFP was exclusively observed in the rnh1∆ rnh2∆ mutants (depicted by an arrow). The novel spot observed in rnh1∆ rnh2∆ cells (adjacent to pause site c, depicted by arrow) corresponds to a RFP in the region between the ARS and 5S rRNA gene, and could possibly correspond to the region rCNS3. rCNS3 was identified in the rDNA (151) as corresponding to a previously identified bidirectional RNA PolII promoter (152). No specific function has been attributed to this element, although it seems to play a key role in the regulation of recombination in the rDNA (151). Another important difference is the lack of pausing site e in rnh1∆ rnh2∆ mutants; according to the work of the Zakian group (153), spot e corresponds to RIs stalled at the
ribosomal ARS. The lack of this pausing site might be explained by the temporal appearance of pausing sites in the RNH- mutants. In rrm3∆ mutants, replication pausing sites coincide with
ongoing DNA synthesis, while rnh1∆ rnh2∆ mutant-dependent pausing sites are restricted to late S/G2, at which time the ribosomal ARS might be devoid of origin interacting proteins that are needed to impair fork progression.
To address whether these bubble-shaped intermediates consist of extensive R-loops, after restriction digestion we performed in vitro RNaseH digestion of the genomic DNA from rnh1∆
rnh2∆ yeast cells at the 105 min time point prior to 2D-gel (Figure 18B). RNaseH would digest regions of RNA hybridized to DNA, and therefore if the bubble-shaped molecules simply corresponded to extended RNA:DNA hybrid regions the bubble arcs should collapse upon RNA degradation. This, however, was not the case, as the bubble-shaped molecules were resistant to in vitro RNaseH treatment (Figure 18B, blue arrows). Furthermore, the sub-Y arcs, consistent with the presence of segments of ssDNA, were equally unaffected by RNaseH treatment (Figure 18B, red arrows). Secondly, we questioned whether such molecules contained a 3’ extendable DNA polymerase substrate. Prior to 2D-gel, RIs were subjected to in
vitro treatment with the exo- Klenow Polymerase, gp32 single-stranded DNA binding protein
Figure 18. A. Characterization of
RFP sites in the NTS region in rnh1∆
rnh2∆ as compared to fob1Δ rrm3Δ
mutant strain. Pausing sites are labelled a-e; black arrow represents
unique RFP site. B. Prior to 2D-gel,
RIs isolated from CPT-treated rnh1∆
rnh2∆ cells at 105 minutes were
subjected to heat or enzymatic treatments as indicated. Blue arrows represent bubble arcs; red arrows represent sub-Y arcs. Schematic representation of species that would migrate as sub-Y arcs (right). 2D-gel analyses shown were performed by Nestor García Rodríguez.
and nucleotides (154) (Figure 18B). The disappearance of bubble-shaped molecules suggests that these intermediates are a substrate for the DNA polymerase and indicates that they contain an extendable 3´ hydroxyl group. Together our results open the possibility that the bubble signals were not made up of an RNA:DNA hybrid, but rather have all of the features of replicating molecules, and thus represent origin-independent replication initiation events.