the efficiency of replication pausing. In this context, it is interesting that the P and M abc regions are present at mat2P and mat3M donor loci that are transcriptionally silenced due to heterochro- matin, yet no imprinting occurs at these loci. We therefore analyzed these loci by 2D-gel electrophoresis and could not observe pausing (Figure 8C). It should be noted that the K-region that separates the two loci replicates early [33], so the loci are replicated in opposite directions, with only mat2P being replicated in the direction where pausing would be expected. However, the data clearly show that the chromatin of a region can affect whether a cis-acting sequence mediates replication barrier activity. Potentially, heterochromatin could be preventing the binding of a trans-acting factor(s) required for replication pausing. Thus, although we do not directly show it, the variance in the intensities of the pause signals observed for the genomic and plasmid M abc could at least be partly due to the differences in chromatin of the regions analyzed. There are no sequence requirements to the imprinted nucleotides or the surrounding region One question that remained was whether there was some kind of sequence recognition at the site of imprinting. In both P and M cells, the imprint is introduced at the junction of the H1 domain within a region predominantly comprising of thymidines (The M Replication Pausing and Imprinting sequence is shown in Figure 9A). A published study has shown that a 6-bp PstI restriction site introduced by substitution at the site of imprinting did not affect the overall level of imprinting [13]. However, characterization of the strain detected a nick with a 59 hydroxyl, a result that is consistent with the hydrolysis of a single ribonucleotide present in the DNA. Separate studies showed that when wild-type DNA was purified under conditions that are known to hydrolyze RNA, a one-nucleotide gap was observed at the site of the imprint [17,20,34]. Since the introduction of the PstI site only led to the mutation of one of the two thymidine residues the imprint consists of, the possibility was raised that there was a sequence specificity to the introduction the ribonucleotides into the DNA. To test this, we substituted the two imprinted thymidine residues with either two adenosines, two guanosines or two cytidines (Figure 9A). None of these substitutions affected the level of imprinting and precise mapping of the position of the imprint in hydrolyzed DNA showed that the mutations did not affect where the imprint was introduced (Figure 9B, 9C). Therefore imprinting process is unaffected by the identity of the imprinted nucleotides. Potentially the sequence of the region surrounding the imprint site may be important for the imprinting process. Substitution of the c section of the abc region, which is immediately centromere- proximal to imprint site, reduced the level of imprinting to approximately 50% (Figure 6). However, the substituted region Figure 9. Substitutions in the region around and of the imprinted nucleotides. (A) The wild-type sequence is given on top. The H1 domain sequences are highlighted with a grey shadow. The position of the inverted repeat that flank the imprint is shown with arrows. Below, the mutant sequences introduced are given. Substituted nucleotides are shown in italics and are underlined. (B) Southern analysis of the strains given in panel A. See Figure 2 legend for method. The position of the DSB products is shown. (C) High-resolution Southern blot of the wild-type and mutant strains carrying substitutions at the imprinted nucleotides. A strand-specific probe that hybridizes to the imprinted strand was used. For detailed explanation of the method, see the Materials and Methods section. The positions of the fragments generated by hydrolysis of the imprint are shown. doi:10.1371/journal.pgen.1001328.g009 PLoS Genetics | www.plosgenetics.org 13 March 2011 | Volume 7 | Issue 3 | e1001328