DNA polymerase α (swi7) and the flap endonuclease fen1 (rad2) act together in the s-phase alkylation damage response in S. pombe

Polymerase α is an essential enzyme mainly mediating Okazaki fragment synthesis during lagging strand replication. A specific point mutation in Schizosaccharomyces pombe polymerase α named swi7-1, abolishes imprinting required for mating-type switching. Here we investigate whether this mutation confers any genome-wide defects. We show that the swi7-1 mutation renders cells hypersensitive to the DNA damaging agents methyl methansulfonate (MMS), hydroxyurea (HU) and UV and incapacitates activation of the intra-S checkpoint in response to DNA damage. In addition we show that, in the swi7-1 background, cells are characterized by an elevated level of repair foci and recombination, indicative of increased genetic instability. Furthermore, we detect novel Swi1-, -Swi3- and Pol α- dependent alkylation damage repair intermediates with mobility on 2D-gel that suggests presence of single-stranded regions. Genetic interaction studies showed that the flap endonuclease Fen1 works in the same pathway as Pol α in terms of alkylation damage response. Fen1 was also required for formation of alkylation- damage specific repair intermediates. We propose a model to explain how Pol α, Swi1, Swi3 and Fen1 might act together to detect and repair alkylation damage during S-phase

Identification of a novel type of spacer element required for imprinting in fission yeast

Asymmetrical segregation of differentiated sister chromatids is thought to be important for cellular differentiation in higher eukaryotes. Similarly, in fission yeast, cellular differentiation involves the asymmetrical segregation of a chromosomal imprint. This imprint has been shown to consist of two ribonucleotides that are incorporated into the DNA during laggingstrand synthesis in response to a replication pause, but the underlying mechanism remains unknown. Here we present key novel discoveries important for unravelling this process. Our data show that cis-acting sequences within the mat1 cassette mediate pausing of replication forks at the proximity of the imprinting site, and the results suggest that this pause dictates specific priming at the position of imprinting in a sequence-independent manner. Also, we identify a novel type of cis-acting spacer region important for the imprinting process that affects where subsequent primers are put down after the replication fork is released from the pause. Thus, our data suggest that the imprint is formed by ligation of a not-fullyprocessed Okazaki fragment to the subsequent fragment. The presented work addresses how differentiated sister chromatids are established during DNA replication through the involvement of replication barriers

The wild-type Schizosaccharomyces pombe mat1 imprint consists of two ribonucleotides

The imprint at the mat1 locus of Schizosaccharomyces pombe acts to initiate the replication-coupled recombination event that underlies mating-type switching. However, the nature of the imprint has been an area of dispute. Two alternative models have been proposed: one stated that the imprint is a nick in the DNA, whereas our data suggested that it consists of one or two ribonucleotides incorporated into the otherwise intact DNA duplex. Here, we verify key predictions of the RNA model by characterization of wild-type genomic DNA purified under conditions known to hydrolyse DNA–RNA–DNA hybrid strands. First, we observe one-nucleotide gap at the hydrolysed DNA, as expected from the presence of two ribonucleotides. Second, using a novel assay based on ligation-mediated PCR, a 3′-terminal ribonucleotide is detected at the hydrolysed imprint. Our observations allow the unification of available data sets characterizing the wild-type imprint

DNA replication : methods and protocols

Since the discovery of DNA structure and throughout the ensuing 'DNA era', the field of DNA replication has expanded to cover a vast number of experimental systems. In "DNA Replication: Methods and Protocols", expert researchers present a collection of techniques and approaches used to investigate DNA replication with an emphasis on the most recent technological developments. Beginning with several informative introductory review chapters, this extensive volume is organized for clarity while fully encouraging innovation by the mixing of methods to create new techniques. Written in the highly successful "Methods in Molecular Biology Series" format, chapters contain brief introductions to the topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, "DNA Replication: Methods and Protocols" provides an excellent tool for both established laboratories and individuals new to this exciting field of research

Characterization of a cis-acting region named <i>abc</i> that is required for replication pausing and imprinting.

<p>(A) Left, line drawing representation of the strains used. A gap represents a deletion and a gray line segment represents a substitution of a DNA sequence. The defined <i>abc</i> region is shown and the borders of the element are indicated by vertical lines. Right, names of the strains and quantification of DSB products and pause signals on 2D gels (including standard error) are given. (B) Southern analysis of the strains shown in panel A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method. (C) 2D-gel analysis of the strains shown in panel A. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method.</p

Southern analysis of <i>mat1</i> replication intermediates.

<p>(A) Line drawing of the fragments generated for different strains for denaturing polyacrylamide gel electrophoresis (PAGE) analysis of replicating <i>mat1M</i> DNA shown in panels B and C. The positions of the restriction enzyme sites and the mapped priming site, and the lengths of the fragments are given. The site of the imprint is indicated with a circle. (B) Denaturing PAGE analysis of the <i>mat1M</i> lagging strand. Replicating DNA from log-phase cells was isolated as for 2D-gel analysis, digested with restriction enzymes given in panel A, then was subjected to denaturing PAGE analysis. After blotting, the membrane was probed with a strand-specific probe that hybridizes to the nascent lagging strand and the parental upper strand. Denatured and labelled DNA ladder is given to the left with size markers. The strains that exhibit imprinting (WT and SS25) produced two strong bands that correspond to the centromere-distal and proximal DSB products created by hydrolysis of the imprint (1d and 1p, respectively). Specific priming products at the site of the imprint (2) migrate to the same position as the distal DSB product observed for the WT and SS25 strains (1d). See panel D. Two fainter putative priming products that are shared between WT and SS25 are marked by arrowheads. (C) Denaturing PAGE analysis of the <i>mat1M</i> leading strand. The nascent leading strand and the parental lower strand were detected by the strand-specific probe. The co-migrating signals from the leading strand intermediates stalled at the imprint in WT and SS25 strains (3) and at <i>MPS1</i> in the strain SS13 (4) are indicated. See panel D. Denatured and labelled DNA ladder is given to the left with size markers. (D) Line drawings of the replication intermediates observed in panel B and C are given. Fragments that produce bands in PAGE analysis are marked. The <i>H1</i> domain is represented as a grey rectangle. The imprint is shown as a black circle. The positions of the restriction enzyme sites used are indicated by vertical lines. (E) 2D-gel analysis of the <i>mat1P</i> and <i>mat1M</i> imprint regions contained in <i>Dra</i>I fragments. Migration of size markers after the first-dimension electrophoresis step is shown below each 2D gel. A line drawing of the region is given on right. The positions of the mapped priming site and the probes are shown. The site of the imprint is indicated with a circle.</p

Transcription of the mating-type-specific gene <i>Mc</i> does not affect imprinting.

<p>(A) The names of the strains and quantification of the DSB products generated during growth in either low-nitrogen sporulation (PMA+) or rich (YEA) media are given. The levels of imprinting, as assessed by DSB products, are similar for both growth conditions. (B) The effect of abolishing <i>Mc</i> transcription on the spacer deletion phenotype. Left, schematic representation of the <i>mat1M</i> cassette and the strains used is given. The positions of the <i>Mi</i> and <i>Mc</i> transcripts are shown by horizontal arrows. Deletion of the promoter region between the two transcripts (in strains SS66 and SS63) was performed, thus abolishing transcription. Right, the names of the strains and quantification of the DSB products (including standard error) are given. (C) Southern analysis of the strains shown in panel B. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method. (D) Northern analysis of total RNA from the strains shown in panel B. The blot was probed for <i>Mc</i> using a double-stranded fragment generated by PCR. (E) Characterization of the strain (SV20) carrying a deletion of most of the spacer region and the region containing the previously mapped priming site. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> legend for method. The position of the deletion is shown as a line drawing. Please note that fork-regression at <i>MPS1</i> is observed in the 2D-gel displayed, in the absence of the imprint. This observation is fully analyzed in a related study (Vengrova and Dalgaard, in preparation).</p

Substitutions in the region around and of the imprinted nucleotides.

<p>(A) The wild-type sequence is given on top. The <i>H1</i> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#pgen-1001328-g002" target="_blank">Figure 2</a> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1001328#s4" target="_blank">Materials and Methods</a> section. The positions of the fragments generated by hydrolysis of the imprint are shown.</p

Working model for <i>MPS1</i> replication pausing and <i>mat1</i> imprinting.

<p>The <i>mat1</i> locus and the events that occur during replication of it are depicted with line drawings. Left, molecular events that take place in the wild-type strain. Right, events occurring when cis-acting mutations are introduced. Positions of the spacer and <i>abc</i> regions and the <i>H1</i> domain (grey boxes) are shown. The orientations of the DNA strands are indicated. The putative proteins mediating <i>MPS1</i> activity and maintenance of the imprint are displayed as a circle and a pentagon, respectively. The imprint is marked with a black spot. Leading- and lagging-strand replication is represented with black half arrowheads. Specific priming is depicted as wavy lines, while loss of it is indicated with grey half arrowheads. Left, positions of the cis-acting deletions are given. See main text for description of each step.</p

Out-competition of the putative trans-acting factor(s) that interacts with the <i>M abc</i> and <i>P abc</i> regions.

<p>(A) The effect of plasmids that carry arrays of the <i>abc</i> region on sporulation efficiency. Two different plasmids (constructed using pREP3X or pREP4X parental vectors) that each carries an array of approximately 10 copies of either the <i>M abc</i> or <i>P abc</i> region were introduced in the <i>h⁹⁰</i> wild-type background (strain JZ1), such that two plasmid-borne arrays of either <i>M</i> or <i>P abc</i> were present. The sporulation efficiency relative to that observed for a strain carrying empty vectors (100%) are given as a graph. The deviation is shown as error bars. (B) Quantification of <i>mat1</i> imprinting in strains used in panel A. <i>Hind</i>III fragments were run and probed with the <i>mat1P Hind</i>III fragment. The intensity of the signal from the <i>P abc</i> array is higher due to greater sequence homology. Quantification of intensities of the DSB products relative to the strain that carries the empty vectors (100%) is given below. (C) Neither <i>P abc</i> nor <i>M abc</i> induces replication pausing at the transcriptionally silenced donor loci. Left, 2D-gel analyses of <i>mat2P</i> and <i>mat3M</i> replication are shown. Right, line drawings of the fragments analyzed are given. The direction of replication through each locus is indicated with grey arrows. The positions of the restriction enzyme sites and the probes used are shown. The sizes of the fragments are given.</p