mat1 locus and the direction-of-replication model. (A) The mating-type region of S. pombe. mat1, containing either P or M information; mat2P and mat3M cassettes are shown. Each cassette is flanked by homology domains H1 and H2 (boxes). The positions of the centromere (circle) and the cis-acting sequences SAS1 and SAS2 (bracket) relative to mat1 are indicated. The recombination event, which replaces the cassette at mat1 with the information of the opposite mating type, is shown by black arrows. The silencing of mat2 and mat3 is indicated by the gray line. (B) The direction-of-replication model. The mating-type pedigree of a newly switched cell is shown. The cells are shown in yellow, and the cell generation is indicated to the right. Next to each cell is a diagram of the sister chromatids, present in the corresponding cell type. The strands, replicated as leading and lagging, are shown as red and blue arrows, respectively. The imprint is indicated by a yellow circle, and the diagram of initiation of the recombination event is displayed in brackets. (C) Replication at the mat1 locus. mat1 replication by a distal origin is secured by a polar replication terminator RTS1, shown as a gray triangle. Nascent leading and lagging strands are indicated by continuous red and discontinuous blue arrows, respectively. Homology domains H1 and H2 are indicated by gray boxes. The white arrows below represent the direction of replication at mat1. The swi1- and swi3-dependent replication pause is shown as a gray shadow.

Leading-strand replication is blocked by the presence of the imprint in the template strand. (A) Mapping of the end of the nascent leading strand at the imprint using high-resolution Southern blot technique. Wild-type (JZ105) and a nonswitching swi3 (SV1) strains are analyzed in parallel. The sequence of the region analyzed is given to the right. The position of the band is indicated by the arrow. (B) Schematic diagram of the analyzed intermediates. The line drawing displays the position of the SspI sites and the size of the analyzed fragment. The imprint is indicated by a circle. The 3′-end of the nascent leading strand, mapped in A, is shown as an empty arrowhead.

Characterization of the mat1 imprint. (A) Primer extension on the intact mat1 imprint allows read-through of the Vent_R(exo–) polymerase, and the RNase T2 treatment converts the imprint into a break. Nonreplicating DNA from JZ105 strain was treated with RNase T2 or NaOH and analyzed by primer extension. The sequence of the region analyzed is given to the right, where the position of the bands is indicated by the arrows. The line drawing below shows the position of the primer used for the analysis relative to the site of the imprint and the interpretation of the obtained result. (B) RNase T2 can recognize one ribonucleotide present in the single-stranded DNA. End-labeled oligonucleotides 1 (without ribonucleotide) and 2 (with one ribonucleotide), shown in D, were treated with RNase T2. The arrow indicates the position of the band generated by RNase treatment. (C) Primer extension using Vent_R(exo–) polymerase on different synthetic templates. The templates, given in D, and the number of cycles used are shown above the autoradiographs. (D) Sequences of the single-stranded templates for primer extension used in B and C. The position where primer priSV2 anneals is shown. The sequence of homology domain H1 is outlined by a gray box. (E) Analysis of the imprint by primer extension on broken DNA, treated with different nucleases. The enzymes used are shown above the respective lanes. The drawing to the right displays the conversion of the imprint into a break by ribonucleases.

Replication pausing at mat1. (A) The summary of the mapping data. The position of the homology domain H1 is indicated by the gray box, and the nucleotides that the imprint consists of are circled. The palindromic bases around the imprint are labeled by arrows, indicating the center of the palindrome (Nielsen and Egel 1989). The start of the smt-0 deletion, extending into the sequence distal to mat1, is shown (Styrkarsdottir et al. 1993). The restriction sites, used in this study for the leading-strand mapping, are labeled, and the positions of the mapped replication intermediates are indicated by horizontal arrows and brackets. (B) The three-step model for the role of pausing in the formation of the imprint (see text). (C) Model for how the imprint can be introduced postreplicatively by oxidation.

2D-gel analysis of the NdeI fragment, containing replication intermediates of mat1. (A) Analysis of the wild-type, SP835 (left), and smt-0, JZ108 (right), strains. The boxes above the autoradiographs show the magnification of the apex region of the Y-arc. The left and the middle correspond to the wild-type, and the right to the smt-0 2D gels. The arrows indicate the position of the cone signal. The line drawings below the autoradiographs show the position of the NdeI sites relative to mat1 and the distance from these sites to the imprint (circle) in wild-type and smt-0 DNA. The position and the length of smt-0 deletion are given. The diagram to the right displays the signals observed on a 2D gel of the wild type. The pause signal and the cone signal are shown, and the “chicken foot” structure, which constitutes the cone signal, is displayed above. The position of the 1N and 2N signals and the distance between them are indicated by the dotted lines and the bracket. The displacement of the cone signal relative to the pause signal is also shown by dotted lines and the small bracket below the diagram. (B) The quantification analysis of the 2D gels of wild-type and smt-0 strains. The diagram displays the signals quantified as well as the structure of corresponding replication intermediates. The intermediates present in switchable and unswitchable cells are shown. The intensities of the ascending (1, light gray) and descending (2, dark gray) parts of the Y-arc of wild type (JZ1) and smt-0 (JZ108) were quantified using Quantity One software from Bio-Rad on three 2D gels for each strain. The signal, labeled 1, was used to normalize the data. The average and standard deviation were calculated for value 2, as displayed on the histogram.

2D-gel analysis of the rqh1 mutant. (A) 2D gels of the NdeI-digested DNA from wild-type (JZ217) and rqh1 (JZ477) strains. The arrows show the position of the cone intermediates. (B) Interpretation of the autoradiographs. The diagram displays the position of the pause signal and the cone signal on the Y-arc, which represents mat1. The “chicken foot” structure, which constitutes the cone signal, is shown. The second Y-arc, which is visualized by the M-specific probe, represents mat3M. The line drawing below shows the position of the NdeI sites relative to mat1 and the distance from these sites to the imprint (circle). The line drawing to the right displays the process of fork reversal at the site of the imprint and the role of rqh1 in resetting reversed forks.

Analysis of replication intermediates at mat1. (A) Primer extension on Okazaki fragments from the wild-type (JZ105), smt-0 (JZ108), swi1 (SV5), swi3 (SV1), and swi7 (SP469) strains. The left panel shows the analysis of the wild-type and smt-0 strains. The position of the homology domain H1 and the site of the imprint are given to the right of the autoradiograph. The drawing below shows the annealing position of the primer used for the assay on the wild-type DNA and lack of specific annealing to smt-0 DNA. The right panel shows the analysis of swi1, swi3, and swi7 mutants. Black lines indicate the position of the analyzed region relative to the left panel. The drawing below indicates that in this experiment the parental DNA, shown by dotted lines, was digested away by λ-exonuclease before primer extension. (B) Mapping of the pause site on the lagging strand on gel-purified paused intermediates. Strains are given in A. The sequence of the region (upper strand) is shown to the right, and the arrows indicate the position of the bands observed. The drawing below shows that RNase H removes RNA primers at 5′-ends of pausing intermediates of the lagging strand. The drawing also indicates that in this experiment primer can anneal both to nascent and to parental DNA. (C) Mapping of the pause site on the leading strand. The nonswitching strain smt-0 (JZ108) was used for clarity of the analysis, to avoid detecting imprinting and switching intermediates. The sequence of the analyzed region is shown to the right. The position of the BstNI restriction site on the upper strand is labeled, and the region of pausing is designated by the brackets. The line drawing below displays the position of the BstNI and SfaNI sites and the size of the full-length fragment of the lower strand. The strands, visualized on the gel, are shown as thick lines, and the mapped 3′-end of the nascent leading strand is shown as an empty arrowhead.