Unbiased segregation of fission yeast chromosome 2 strands to daughter cells

Abstract

The base complementarity feature (Watson and Crick in Nature 171(4356):737-738, 1953) and the rule of semi-conservative mode of DNA replication (Messelson and Stahl in Proc Natl Acad Sci U S A 44:671-682, 1958) dictate that two identical replicas of the parental chromosome are produced during replication. In principle, the inherent strand sequence differences could generate nonequivalent daughter chromosome replicas if one of the two strands were epigenetically imprinted during replication to effect silencing/expression of developmentally important genes. Indeed, inheritance of such a strand- and site-specific imprint confers developmental asymmetry to fission yeast sister cells by a phenomenon called mating/cell-type switching. Curiously, location of DNA strands with respect to each other at the centromere is fixed, and as a result, their selected segregation to specific sister chromatid copies occurs in eukaryotic cells. The yeast system provides a unique opportunity to determine the significance of such biased strand distribution to sister chromatids. We determined whether the cylindrical-shaped yeast cell distributes the specific chromosomal strand to the same cellular pole in successive cycles of cell division. By observing the pattern of recurrent mating-type switching in progenies of individual cells by microscopic analyses, we found that chromosome 2 strands are distributed by the random mode in successive cell divisions. We also exploited unusual "hotspot" recombination features of this system to investigate whether there is selective segregation of strands such that oldest Watson-containing strands co-segregate in the diploid cell at mitosis. Our data suggests that chromosome 2 strands are segregated independently to those of the homologous chromosome.

Fig. 1

a The S. pombe mat1-switching system (figure modified from Klar 2007). The mat locus spans about 30 kbp in the middle of chromosome 2. The cell-type determining mat1 locus is switched between two allelic forms by transposing a copy of genetic information, residing in the mat2 or mat3 donor locus, through substitution of the resident mat1allele. The short H1 and H2 “homology boxes” present in all cassettes are used to transfer about 1.1 kbp allele-specific sequences copied from donor loci by recombination to produce a switched mat1 allele. The location of the imprint is indicated with a blue-colored star. b The DNA strand-specific imprint/segregation mechanism of mat1 switching. The two consecutive asymmetric cell divisions in the cell pedigree are due to inheritance of specific parental chromosomal strands by the specific daughter cell such that only one in four granddaughter cells switches. The “Watson” (W) strand is drawn at the top and the “Crick” strand at the bottom. Only the W strand synthesized by the lagging-strand replication complex is imprinted at mat1 and it is shown by a green-colored line to help follow its distribution in the pedigree. The switched mat1-P allele is presented in red color, whose both strands are synthesized de novo by the mat1-switching mechanism. Details are described in the text of the paper

Fig. 2

Diagrams of three hypothetic modes of strand distribution to specific poles of the parental cell. The pedigree is conducted with diploid SP10 (h⁹⁰/mat1:3M) cells permitting us to score sporulation ability of individual cells. After mat1-M to mat1-P switch had occurred in the chromosome containing the mat1-M imprinted locus, equivalent to the Ms allele in Fig. 1b, the recently switched cell with the mat1-P allele would form an ascus. Segregation mode of a specific strand of one of the chromosome 2 copies was identified by microscopically observing ascus formation by the progenies of single cells

Fig. 3

The chromosome 2 arms swapping hypothesis. It is advanced to explain the recombination mechanism of the mat1 hotspot. The location of genetic markers is not drawn to scale. The meiotic genetic distance between leu1 and mat1 markers is 10 cM, mat1 and his2 is 2.0 cM, and between mat1 and ade1 is >50 cM. According to our arms-swapping hypothesis, recombination occurs only between non-sister chromatid #1 with #3 and only when they simultaneously have DNA breaks (\cr) in the S/G2 phase of the cell. To depict specific strand distribution, template Watson (W) strands are colored green and Crick (C) ones are colored red. Strands synthesized in the present replication cycle are depicted in black color. The symbol X represents the crossover point at mat1. The numbers 1 to 4 represent chromatid number assigned to their centromeres. Two classes of recombinants concerning genetic markers flanking mat1 are shown. The 1+4 and 2+3 chromatid segregation, where each daughter cell inherits a single recombined chromatid, causes homozygosis of all markers distal to the crossover point. When both recombined chromatids 1+3 co-segregate to one of the daughter cells, it remains heterozygous for all the markers, but notably, coupling of flanking markers changes due to recombination

Fig. 4

Homozygosis experiment. The procedure tests phenotype of colonies seeded with sister cell pairs after their growth on rich (YEA) medium. The adjacent colonies in the picture represent those seeded with sister cell pairs. These colonies were scored for his2 and ade1 markers by replica plating them to appropriate drop-out media

Fig. 5

Classes of recombination due to hotspot. Homozygosis frequency and markers map distance in strain SP1146 subclones is reported. The values of meiotic genetic distance from the literature of markers of conventional strains without hotspot recombination are indicated at the top. Results of tetrad analysis are tabulated. PD parental ditype, NPD non-parental ditype, TT tetra-type tetrads, cM the apparent meiotic map distance calculated in centimorgans, cM* adjusted meiotic map distance calculated by deducting the contribution of hotspot recombination