This study characterized temperature-sensitive mutants of the DNA polymerase gene polb in the fission yeast Schizosaccharomyces pombe. The researchers generated three conditional lethal polb mutant alleles through random mutagenesis. At the restrictive temperature, all three mutants arrested in S phase of the cell cycle and exhibited a typical cell division cycle terminal phenotype. Sequencing revealed that the temperature-sensitive mutations were missense mutations altering amino acids uniquely conserved among known polymerase 6 sequences.

1. Nucleic Acids Research, 1993, Vol. 21, No. 16 3821 -3828
Fission yeast with DNA polymerase 6 temperature-sensitive
alleles exhibits cell division cycle phenotype
Stefania Francesconi, Hyunsun Park and Teresa S.-F.Wang*
Laboratory of Experimental Oncology, Department of Pathology and Department of Pharmacology,
Stanford University School of Medicine, Stanford, CA 94305-5324, USA
Received March 30, 1993; Revised and Accepted June 29, 1993
ABSTRACT
DNA polymerases a and 6 are essential enzymes
believed to play critical roles in initiation and replication
of chromosome DNA. In this study, we show that the
genes for Schizosaccharomyces pombe (S.pombe)
DNA polymerase a and 6 (pola + and pol0+) are
essential for cell viability. Disruption of either the
pola+ or polb+ gene results in distinct terminal pheno-
types. The S.pombe polb+ gene is able to complement
the thermosensitive cdc2-2 allele of Saccharomyces
cerevisiae (S.cerevisiae) at the restrictive temperature.
By random mutagenesis in vitro, we generated three
polb conditional lethal alleles. We replaced the wild type
chromosomal copy of polb+ gene with the muta-
genized sequence and characterized the thermosensi-
tive alleles in vivo. All three thermosensitive mutants
exhibit a typical cell division cycle (cdc) terminal
phenotype similar to that of the disrupted polb+ gene.
Flow cytometric analysis showed that at the nonper-
missive temperature all three mutants were arrested in
S phase of the cell cycle. The three S.pombe con-
ditional polb alleles were recovered and sequenced.
The mutations causing the thermosensitive phenotype
are missense mutations. The altered amino acid
residues are uniquely conserved among the known
polymerase 6 sequences.
INTRODUCTION
Two major events of the cell cycle are DNA replication and
mitosis. There are multiple feedback mechanisms to ensure the
completion of DNA replication, DNA repair and chromosome
segregation before cell division (1). Mutants that uncouple mitosis
from the completion of DNA replication have been isolated in
S.cerevisiae and S.pombe (2-5). To unravel the mechanisms
regulating S phase, and to pave the way for understanding the
feedback control mechanisms of cell cycle dependency, we
studied the fission yeast DNA polymerase genes. We isolated
the genes for S.pombe DNA polymerases a and 6 (pola+ and
polb+) and studied their expression during the cell cycle (6). In
this report, we describe the generation ofthree conditional lethal
alleles of polb gene. At restrictive temperature, these mutants
exhibit typical cell cycle division (cdc) terminal phenotype and
arrest specifically in the S phase of the cell cycle.
MATERIALS AND METHODS
Yeast and E.coli strains and media
The following S.pombe strains were used in this study: Two
diploid strains SP826 (h+/h+ ura4-D18/ura4-D 18
leul-32/leul-32 ade6-M210/ade6-M216) (4) and SP817
(h+N/h-S ura4-D18/ura4-D18 ade6-M210/ade6-M216) were
used for gene disruption. The haploid strain, SP808, (h+N
ura4-D-18 leul-32 ade6-M216), was used for isolating the
thermosensitive mutants. The cdc2 and cdclO haploid strains
carrying the cdc2-33 and cdc10-129 alleles were used as cell cycle
markers for flow cytometric analysis (7). The diploid and haploid
strains used in this study were from David Beach (Cold Spring
Harbor) and Robert Booher (University of California at San
Francisco). Strains ED612 (cdc23-M36 leul-32 h+) and ED613
(cdc2l-M68 leul-32 h+) (8) were from P.Fantes (University of
Edinburg). Strain EDSF087 (cdc20-MIO leul-32 h+) was
produced by crossing strain ED087 (cdc20-M1O h+) from
P.Fantes with strain SP812 (leu 1-32 ura4-D18 ade6-M210
h-S). The S.cerevisiae strain 9048-13-2 (cdc2-2 pep4 prbl
ura3-52 his7 cani) (9) used for the complementation experiment
was from A. Morrison (NIEHS).
The E.coli JM105 strain was used for plasmid constructions.
The JA226 E. coli strain was used to recover the plasmid from
genomic DNA from Spombe transformants. Spombe was grown
either in YEA or EMM media supplemented with essential amino
acids and appropriate supplement (10,11). S.cerevisiae was grown
in either YPD or SD media supplemented with essential amino
acids and appropriate supplement. Medium containing 5-FOA
was formulated as described in (12).
Plasmids
To construct the plasmid YEpPolb for complementation of the
S. cerevisiae cdc2-2 allele, a 3280 bp BamHI DNA fragment of
polb+ containing the full-length coding sequence of the gene and
a deletion ofthe 52 bp short intron (6) was inserted into the unique
BglIH site of the ADHI promoter of YEpADH. The YEpADH
plasmid is a derivative ofYEp24 with the ADH1 promoter cloned
* To whom correspondence should be addressed
.=) 1993 Oxford University Press