Mol. Cells, Vol. 12, No. 3, pp. 277-285
Molecules
and
Cells
Minireview
KSMCB 2001
Yeast Replicative DNA Polymerases and Their Role at the
Replication Fork
Yasuo Kawasaki and Akio Sugino*
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
(Received August 7, 2001; Accepted August 14, 2001)
The budding yeast, Saccharomyces cerevisiae, is an
excellent model system for the study of DNA polymerases and their roles in DNA replication, repair,
and recombination. Presently ten DNA polymerases
have been purified and characterized from S. cerevisiae. Rapid advances in genome sequencing projects
for yeast and other organisms have greatly facilitated
and accelerated the identification of yeast enzymes
and their homologues in other eukaryotic species. This
article reviews current available research on yeast
DNA polymerases and their functional roles in DNA
metabolism. Relevant information about eukaryotic
homologues of these enzymes will also be discussed.
Keywords: DNA Polymerase; DNA Replication; Replication Fork; Saccharomyces cerevisiae; Three DNA Polymerase Model.
Introduction
DNA polymerases are DNA-dependent nucleotidyltransferases that replicate double-stranded DNA in a semiconservative manner. DNA polymerases are required to
duplicate the genetic material prior to cell division. These
enzymes are also required for DNA repair, recombination,
and translesion DNA synthesis to preserve genomic integrity.
The discovery of the first eukaryotic DNA polymerase,
DNA polymerase α (Pol α), was in 1957. This was approximately one year after DNA polymerase I was isolated from Escherichia coli. DNA polymerases β and γ
were identified in the early ’70s. DNA polymerases δ and
ε were discovered in the ’80s. From the late ’90s until the
present, a large number of DNA polymerases that may be
* To whom correspondence should be addressed.
Tel: 81-6-6879-8331; Fax: 81-6-6877-3584
E-mail: [email protected]
involved in translesion DNA synthesis were identified in
many organisms from bacteria to humans. The discovery
of these polymerases greatly promoted our understanding
of the biological function(s) of DNA polymerases in DNA
replication, repair, and recombination.
It is generally accepted that replicative DNA polymerases are essential for cell viability, but that DNA polymerases that are involved in other biological processes
may be dispensable for cell growth. The replicative DNA
polymerases α, δ, and ε are required for chromosomal
DNA replication. They are also essential for cell growth
in yeast. However, two other DNA polymerases, Pol σ
and φ, are also essential for cell viability. Because eukaryotic genomes are larger and more complex than prokaryotic genomes, more DNA polymerases are needed to
maintain the integrity of the genomes in eukaryotes. Recently, a variety of DNA polymerases have been discovered that may be involved in translesion DNA synthesis.
Some of these synthesize DNA in an error-free manner,
others synthesize DNA in an error-prone manner. Translesion DNA synthesis is an important process that allows
the replication fork to bypass DNA lesions that are not
removed prior to the S phase. The translesion DNA polymerases are conserved from bacteria to humans, suggesting that they may play an important, although incompletely characterized, role in maintaining genomic stability.
In the yeast Saccharomyces cerevisiae, a variety of biochemical and genetic approaches have been used to investigate the function of DNA polymerases. In addition,
structural and functional homologue(s) for each yeast
DNA polymerase have been identified in other eukaryotes.
This review summarizes current information on the yeast
replicative DNA polymerases, and discusses their roles at
the replication fork. Newly identified essential DNA polymerases Pol σ and φ will also be discussed.
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The Yeast DNA Polymerases
Replicative DNA polymerases
DNA polymerase α (I) DNA polymerase α (I) is a complex of four polypeptides (Lucchini et al., 1985). Each is
encoded by an essential gene (Table 1, Campbell and
Newlon, 1991; Campbell, 1993). The largest of these
polypeptides is the catalytic subunit. The 86 kDa polypeptide’s function is unknown. The remaining two polypeptides are both required for the polymerase α-associated
primase activity (Santocanale et al., 1993). DNA polymerase α is the only DNA polymerase that can initiate
DNA synthesis on a single-stranded DNA template. Initially, some reports suggested that exonuclease activity is
associated with DNA polymerase α; however, evidence
now conclusively shows that eukaryotic DNA polymerase
α has no exonuclease activity (Burgers, 1999).
DNA polymerase α-primase has been characterized
extensively using the in vitro SV40 DNA replication system. In this system, DNA polymerase α-primase initiates
synthesis of the leading and lagging strands at the initiating replication fork (Waga and Stillman, 1994). It was
therefore predicted that little or no DNA synthesis would
occur at the restrictive temperature in yeast cells that
carry conditional lethal mutants of pol1 or pri (primasenegative) (Campbell, 1993; Foiani et al., 1989). In
contrast, although chromosomal DNA synthesis is
impaired in these mutants, DNA synthesis still proceeds,
in these mutants, DNA synthesis still proceeds, even
when cells are transferred to the restrictive temperature
during G1 (Budd et al., 1989; Lucchini et al., 1988). A
much stronger inhibition of DNA synthesis is observed at
the restrictive temperature when these mutants are used in
the in vitro SV40 DNA replication system. One possible
explanation for this result is that another DNA polymerase might elongate DNA that is initiated in vivo by
residual DNA polymerase α activity.
The mutant DNA polymerase α that is encoded by pol1
(cdc17) has been well characterized. This mutant displays
several phenotypes. These include the following: impaired DNA replication (Foiani et al., 1994), enhanced
rates of mitotic recombination, gene conversion, and excision of a large inverted region; increased spontaneous
mutation rate; sensitivity to alkylating agents; increased
telomere length; and polyploidy (Foiani et al., 1994). Because the second largest subunit of DNA polymerase α is
phosphorylated early in the S phase and then dephosphorylated as the cells exit from mitosis, this subunit
might play a role in regulating the initiation of DNA synthesis (Foiani et al., 1995).
DNA polymerase ε (II) Yeast DNA polymerase ε (II)
was identified at the same time as yeast DNA polymerase
α (Chang, 1977; Wintersberger, 1974; Wintersberger and
Table 1. DNA polymerases in Saccharomyces cerevisiae.
DNA
Molecular mass
polymerase of subunit (kDa)
Gene
Essential?
Function
Homologue
α (I)
180
86
58
48
POL1 (CDC17)
POL10
PRI1
PRI2
Yes
Yes
Yes
Yes
Chromosomal DNA replication
DNA polymerase α
ε (II)
256
80
34
31
29
POL2
DPB2
DPB3
DPB3
DPB4
Yes
Yes
No
No
No
Chromosomal DNA replication
and repair
DNA polymerase ε
δ (III)
125
48
55
POL3 (CDC2)
POL31 (HYS2)
POL32
Yes
Yes
No
Chromosomal DNA replication
and repair
DNA polymerase δ
β (IV)
68
POL4
No
DNA polymerase β
φ (V)
116
POL5
Yes
Double-strand break repair
(Base excision repair?)
rRNA synthesis?
σ
66
72
TRF4
TRF5
Yes*
Sister chromatid cohesion
Homologues in other
eukaryotes
γ
144
MIP1
No
Mitochondrial DNA replication
DNA polymerase γ
ζ
173
REV3
REV7
No
No
Error-prone translesion DNA synthesis
DNA polymerase ζ
η
71
RAD30
No
Error-free translesion DNA synthesis
Rev1
112
REV1
No
Deoxycytidyl transferase Translesion
DNA synthesis
DNA polymerase η, ι, κ
UmuC, DinB in E. coli
Rev1
* Both TRF4 and TRF5 encode DNA polymerase σ and deletion of both genes causes loss of viability (see text).
Pol5 in S. pombe
Yasuo Kawasaki & Akio Sugino
Wintersberger, 1970). DNA polymerase ε activity was
described in mammalian cells in 1985 (Byrnes, 1985),
although the activity was initially thought to be related to
DNA polymerase δ. Early studies also showed that DNA
polymerase ε activity was higher in the cells in the stationary-phase than in cells undergoing logarithmicgrowth, suggesting that it might function in DNA repair
(Chang, 1977). DNA polymerase ε was not studied extensively until 1990, when two groups characterized its
activity (Budd et al., 1989; Hamatake et al., 1990). Two
forms of DNA polymerase ε were found in S. cerevisiae.
The first identified form of DNA polymerase ε has an
apparent molecular mass of approximately 145 kDa on a
SDS-polyacrylamide gel (Hamatake et al., 1990). The
second identified form of DNA polymerase ε has five
polypeptides (holoenzyme). Their molecular masses are
from 29 to 256 kDa (Hamatake et al., 1990). Partial proteolysis studies demonstrated that the 145 kDa polypeptide is a degraded form of the 256 kDa polypeptide. Both
the 145 kDa and holoenzyme forms of DNA polymerase ε
have 3′ to 5′ exonuclease activity, which removes any
error that could be made during DNA replication (Morrison and Sugino, 1994; Morrison et al., 1993). The biochemical properties of DNA polymerase ε are quite similar to those of DNA polymerase δ (see below), although
DNA polymerase ε is more processive than DNA polymerase δ in the absence of co-factors. The genes that encode all four subunits of DNA polymerase ε have been
cloned and sequenced (Araki et al., 1991; 1992; Morrison
et al., 1990; Ohya et al., 2000). The genes for the largest
and second largest subunits, POL2 and DPB2 respectively,
are essential for cell growth in S. cerevisiae, whereas
DPB3 and DPB4 are not essential.
Several conditionally lethal mutants of POL2 have been
isolated and characterized. In these mutants, chromosomal
DNA synthesis ceases at the restrictive temperature, indicating that DNA polymerase ε is required for normal
chromosomal DNA replication. The terminal morphology
of these mutant cells is a dumbbell shape with a single
nucleus that is localized between the mother and daughter
cells. This is the same terminal morphology of other DNA
replication mutants in S. cerevisiae. Furthermore, a temperature-sensitive mutant of the DPB2 gene, which encodes the second largest subunit of the DNA polymerase ε
holoenzyme, results in temperature sensitive chromosomal DNA replication (Araki et al., 1992). These, included
with other results, do not support a direct and/or primary
role for DNA polymerase ε in DNA repair. For example,
several mutant pol2 alleles have temperature-sensitive
DNA polymerase ε activity in vitro, but show no significant sensitivity to DNA-damaging reagents in vivo. Thus,
although a role for DNA repair is not completely excluded (see below), it is likely that other DNA polymerases are important for DNA repair in yeast (Araki et
al., 1992).
279
When assayed using an in vitro DNA repair system,
crude extracts from temperature sensitive pol2 mutant
cells catalyze a temperature sensitive DNA repair reaction
(Wang et al., 1993). Furthermore, it has been suggested
that in human cells, DNA polymerase ε is involved in
DNA repair processes that are initiated by UV-damage
(Nishida et al., 1988). Generally, DNA damage induces
the expression of many genes that may enhance the DNA
repair capacity of the cell. Several DNA damageuninducible mutations have been isolated and characterized (Zhou and Elledge, 1993). One of these mutations,
dun2, is allelic to pol2, and creates a mutation in the carboxyl-terminal region of the DNA polymerase ε polypeptide. This suggests that DNA polymerase ε may act as a
DNA-damage ‘sensor’ that sends a signal, which increases the expression of DNA repair genes (Navas et al.,
1995). Another study strongly suggests that DNA polymerase ε also participates in an S phase cell-cycle checkpoint (Araki et al., 1995). A multicopy suppressor gene
(DPB11) of the temperature-sensitive dpb2 mutant has
been characterized. It has significant homology to S.
pombe cut5+/rad4+, which is thought to play a role in
DNA synthesis and mitosis (Saka et al., 1994). Dpb11p
physically interacts with DNA polymerase ε and this interaction is limited to the S phase (Masumoto et al., 2000).
In temperature-sensitive dpb11-1 cells, chromosomal
DNA synthesis is inhibited at the restrictive temperature
but, unlike other DNA synthesis mutants, the cell cycle
proceeds and causes rapid cell death (Araki et al., 1995).
This suggests that DPB11 may act positively in the S
phase to promote DNA replication and cell growth, and
negatively at G2-M to prevent cell death. RAD9 and
MEC1-3 genes participate in cell-cycle checkpoints
(Weinert et al., 1994). However, the DPB11 pathway
seems to be completely different than these pathways.
DNA polymerase ε dissociates from the template DNA in
the presence of single-stranded DNA in vitro. This suggests that DNA polymerase ε itself might sense unreplicated DNA (Maki et al., 1998).
Recently, it was reported that an internal deletion of
POL2 inactivates the DNA polymerase, and the 3′ to 5′
exonuclease activities of DNA polymerase ε complements
the lethality of a pol2 deletion strain. In contrast, strains
that carry mutations that affect the carboxyl-terminus of
the enzyme are inviable (Dua et al., 1999; Kesti et al.,
1999). The polymerase/exonuclease deficient deletion
mutant is temperature sensitive for cell growth, exhibits a
slow growth phenotype even at permissive temperatures,
and elongates replicating DNA at a slower rate than wild
type cells. Furthermore, the life span of the mother cell
with this mutation is much shorter than the life span of a
wild type cell. These results indicate that chromosomal
DNA replication can proceed without an active DNA polymerase ε. However, deficiency of this enzyme causes a
cellular defect in the mother cell that may decrease the
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The Yeast DNA Polymerases
integrity of DNA that is replicated in that cell, and therefore decreases the viability of the daughter cells (Ohya,
Kawasaki, Hiraga, Nakajoh, Kanbara, Nakashima, and
Sugino, manuscript submitted for publication). It is possible that DNA polymerase ε plays roles in DNA replication, repair, and recombination. Human DNA polymerase
ε and/or δ are required for gap-filling DNA synthesis during in vitro reconstituted nucleotide excision repair
(Aboussekhra et al., 1995). In a yeast repair assay with a
soluble extract, DNA polymerase ε, rather than DNA polymerase δ, is required for base excision repair (Wang et
al., 1993). In addition, recombinational repair requires
both DNA polymerase δ and ε to repair a double-strand
DNA break (Holmes and Haber, 1999).
DNA polymerase δ (III) DNA polymerase δ (III) is a
moderately processive enzyme that is stimulated by proliferating cell nuclear antigen (PCNA). This characteristic
distinguishes DNA polymerase δ from DNA polymerase ε.
Purified DNA polymerase δ has three polypeptide subunits that migrate as 125, 55, and 50 kDa polypeptides on
SDS-polyacrylamide gels (Bauer et al., 1988; Gerik et al.,
1998; Hamatake et al., 1990). In S. cerevisiae, these polypeptides are encoded by POL3/CDC2, POL31/HYS2, and
POL32, respectively. The enzyme co-purifies with a
tightly associated 3′ to 5′ exonuclease activity that performs an editing function during chromosomal DNA replication (Morrison et al., 1991; Simon et al., 1991). Pol3p
is the catalytic subunit. It is essential for cell growth.
Pol31p is the second largest subunit of DNA polymerase
δ, and it is also essential. In contrast, Pol32p is not essential for cell growth, although ∆pol32 cells have a growth
defect at high temperature, and are sensitive to DNA
damaging agents. DNA polymerase δ that lacks the
Pol32p subunit binds PCNA, but it is not as processive as
the wild type enzyme in the presence of PCNA in vitro
(Burgers and Gerik, 1998). The subunit structure of S.
cerevisiae Polδ is conserved from yeast to humans
(Hughes et al., 1999). Highly purified DNA polymerase δ
from S. pombe is composed of four polypeptides - Pol3p,
Cdc1p, Cdc27p, and Cdm1p (Zuo et al., 1997). Cdc27p, a
homologue of Pol32p, is essential for cell growth in S.
pombe (Zuo et al., 2000), although Pol32p is dispensable
for growth in S. cerevisiae.
PCNA and the structure specific DNA-dependent ATPase RF-C are accessory proteins for Polδ. Three molecules of PCNA form a ring structure called a “sliding
clamp”. It tethers DNA polymerase δ to the template
DNA. RF-C is considered to be a “clamp loader” and is
functionally homologous to gp44 from phage T4, E. coli
DNA polymerase III holoenzyme subunits γ/τ and δ’, and
B. subtilis DnaH.
The cdc2/pol3 mutants have a complex phenotype.
Unlike DNA polymerases α and ε, it is unclear if DNA
polymerase δ is required for chromosomal DNA replica-
tion. The cdc2 mutants have a cell-cycle arrest phenotype
at the restrictive temperature, and although chromosomal
DNA replication does not go to completion, about 70% of
the genome appears to be synthesized after a G1synchronized cell culture is shifted to the restrictive temperature (Conrad and Newlon, 1983). This suggests that
there might be a defect in assembly of the replication
complex that stops the cell from entering a round of replication. However, these explanations are inconsistent with
the model that is based on the reconstituted SV40 in vitro
DNA replication system (Waga and Stillman, 1999).
There, DNA polymerase δ synthesizes both the leading
and lagging strands. Another explanation might be that
the 70% partial completion of DNA synthesis in cdc2
mutants may be due to the residual function of the mutant
DNA polymerase δ. It also may be due to DNA synthesis
by DNA polymerase α or ε in the absence of DNA polymerase δ.
Two additional alleles of the cdc2 mutation, hpr6
(Aguilera and Klein, 1988) and tex1 (Gordenin et al.,
1992), were identified during a search for strains with
high frequency excision of inverted repeat sequences. In
addition to a high frequency of recombination, hpr6 mutants are sensitive to methylmethane sulfonate (MMS)
and resistant to UV-induced lesions at semi-permissive
temperatures. This suggests that DNA polymerase δ is
involved in recombinational repair and DNA replication.
As mentioned previously, DNA polymerase δ also participates in DNA repair. However, the best evidence that
DNA polymerase δ plays a role in elongation during
chromosomal DNA replication comes from experiments
with temperature-sensitive mutants of the S. pombe homologue of DNA polymerase δ (Francesconi et al., 1993).
Distinct roles of replicative DNA polymerases at the replication fork
DNA replication in S. cerevisiae requires at least DNA
polymerases α, δ and ε. Using the in vitro SV40 DNA
replication system, DNA replication is reconstituted with
purified DNA polymerases α and δ. Based on this result,
it was proposed that DNA polymerase α and δ can synthesize both the leading and lagging strands in other eukaryotes (Waga and Stillman, 1994). However, it remained unclear which DNA polymerase synthesized the
leading strand, and which DNA polymerase synthesized
the lagging strand in vivo. Since DNA polymerase ε is the
most processive yeast DNA polymerase, we suggest that
it may synthesize the leading strand, while the Polαprimase complex and Polδ may synthesize the lagging
strand (Fig. 2). However, Burgers analyzed the saltsensitivity of DNA synthesis by DNA polymerases α, δ,
and ε using singly-primed single-stranded DNA in the
presence of PCNA, RF-C, and RF-A proteins. Based on
Yasuo Kawasaki & Akio Sugino
281
Fig. 1. B family DNA polymerases in S. cerevisiae. Closed boxes indicate the DNA polymerase domain that is conserved in B family
DNA polymerases. A blue box indicates the exonuclease domain. A red box indicates a cysteine rich domain that may form a zinc
finger.
his results, Burgers suggested that the Polα-primase complex and Polε are likely to be involved in the lagging
strand synthesis, while Polδ is likely to synthesize the
leading strand. Another group suggests that Polε may participate in the gap-filling reaction that joins two Okazaki
fragments that no longer have their RNA primers. It is
important to state the caveat that these models are exclusively based on data from in vitro experiments. There is
presently no direct data available regarding the strand
specificity of DNA polymerases during DNA replication
in vivo. However, some evidence regarding in vivo polymerase strand specificity was obtained with 3′ to 5′ exonuclease deficient mutants of Polδ and Polε. These studies indicate that Polδ synthesizes one of the two strands of
DNA at the replication fork, and Polε synthesizes the
other strand (Morrison and Sugino, 1994; Shcherbakova
and Pavlov, 1996). Furthermore, Bae et al. (2001) recently showed that Dna2p, Fen1p, and Polδ participate in
a coordinated reaction to mature RNA-linked Okazaki
fragments. This result supports the model in which Polδ
synthesizes the lagging strand at the replication fork (Bae
et al., 2001). Furthermore, it was recently shown that the
immuno-depletion of DNA polymerase ε from Xenopus
egg extracts drastically reduces DNA replication activity
in an in vitro DNA replication system (Waga et al., 2001).
Previously, an important role for DNA polymerase ε in
DNA replication had only been demonstrated in yeast.
Other essential DNA polymerases
DNA polymerase σ TRF4 was identified in a screen for
mutations in S. cerevisiae, which are synthetically lethal
with mutations in yeast DNA topoisomerase I (top1)
(Sadoff et al., 1995). Both TRF4 and its redundant homologue TRF5 encode DNA polymerase σ. Trf4p and Trf5p
have a 55% identity and 72% similarity in their predicted
amino acid sequences (Castano et al., 1996). DNA polymerase σ possesses a DNA polymerase-β type deoxynucleotidyl transferase domain (Aravind and Koonin, 1999).
Purified Trf4p/DNA polymerase σ has a distributive DNA
polymerase activity with low processivity (Wang et al.,
2000). Although initially reported as ‘Polκ’ in the literature (Wang et al., 2000), it was renamed ‘Polσ’, since one
of the human translesion DNA polymerase was previously
named ‘Polκ’ (Ohashi et al., 2000). Polσ is essential for
cell viability in yeast, since trf4 trf5 double-deletion mutants are inviable. Temperature-sensitive trf4-ts trf5 mutants are deficient in sister chromatid cohesion, and in
progression through the S phase at non-permissive temperatures. This suggests that Polσ plays important roles in
coordinating DNA synthesis and in sister chromatid cohesion. A trf4-polymerase-negative mutant with an amino
acid substitution in the polymerase domain does not complement the inviability of the trf4 trf5 double-mutant cells.
This result suggests that the polymerase activity of Polσ
is required for its essential functions (Wang et al., 2000).
However, it is unclear what precise role is played by Polσ
during chromosomal DNA replication in yeast.
DNA polymerase φ Recently we identified the openreading frame of YEL055C, which is homologous to the
conserved DNA polymerase B family motif (Fig. 1). This
gene encodes the fifth essential DNA polymerase in S.
cerevisiae (Winzeler et al., 1999). Purified from yeast cell
extracts, the enzyme is an aphidicolin-sensitive DNA polymerase that is stimulated by PCNA (Pol30p). Thus, we
named it DNA polymerase φ (Shimizu, Hiraga, Kawasaki,
Tawaramoto, Nakashima, and Sugino, manuscript submitted). Temperature-sensitive pol5 mutants were generated
and characterized. The pol5 mutants do not arrest at G2/M
at the restrictive temperature. Furthermore, DNA polymerase-active-site mutants fully complement the lethality
of ∆pol5. This suggests that the DNA polymerase activity
of Pol5p is not required for its essential function in cell
proliferation.
Cytological studies indicate that a large portion of Pol5p
co-localizes with Nop1p in the nucleolus. Total RNA synthesis was severely inhibited in the temperature-sensitive
pol5 mutant cells at the restrictive temperature. This would
282
The Yeast DNA Polymerases
clease activity (Vanderstraeten et al., 1998) and is localized in the mitochondrial matrix. It is clear that DNA polymerase γ is the major enzyme that synthesizes mitochondrial DNA.
Translesion DNA synthesis polymerases:
DNA polymerases η, ξ, and Rev1
Fig. 2. Model for chromosomal DNA replication by DNA polymerases α, δ, and ε. (1) DNA polymerase α-primase initiates
synthesis of a short DNA fragment (blue arrow) that is primed
by RNA (blue zigzag line) at the replication origin. (2) The
DNA fragment is elongated by DNA polymerase ε (leading
strand; red arrow) or by DNA polymerase δ (lagging strand;
green arrow). (3) Progression of leading strand synthesis and
discontinuous lagging strand synthesis. (4) DNA polymerase δ
carries out strand displacement of an RNA-primed Okazaki
fragment that produces a flap structure which is recognized by
Dna2/Fen1 (see Bae et al., 2001). (5) The RNA primer and a
portion of the Okazaki fragment are removed, and the lagging
strand synthesis is completed by DNA polymerases δ and DNA
ligase I (encoded by CDC9).
indicate that Pol5p functions in stable RNA synthesis,
which is consistent with its subcellular localization in the
nucleolus. A homologous gene (pol5+) was identified in S.
pombe, suggesting that DNA polymerase φ may exist in
other eukaryotic cells.
Nonessential DNA polymerases
DNA polymerase β (IV) Yeast DNA polymerase β (IV)
has significant homology to mammalian DNA polymerase
β (Aravind and Koonin, 1999; Prasad et al., 1993; Shimizu et al., 1993). However, the 68 kDa catalytic polypeptide is considerably larger than mammalian DNA polymerase β (39 kDa). Although mammalian DNA polymerase β participates in base excision repair (BER), there
is no evidence that yeast DNA polymerase β participates
in BER. Rather, it appears to be involved in double-strand
DNA break repair (Leem et al., 1994).
DNA polymerase γ DNA polymerase γ is encoded by a
nuclear gene MIP1. Mutants of MIP1 have a defect in
mitochondrial, but not nuclear DNA synthesis (Berger
and Yaffe, 2000). DNA polymerase γ has 3′ to 5′ exonu-
When the replication fork encounters a DNA lesion, such
as an UV-photoproduct, abasic site, oxidized or deaminated base, DNA synthesis by replicative DNA polymerases is blocked at the site of the lesion. In this situation, additional proteins are required to repair or to bypass
these lesions. In eukaryotes, several DNA polymerases
participate in lesion bypass. DNA polymerase η, encoded
by RAD30 in S. cerevisiae, is homologous to UmuC and
DinB in E. coli (Johnson et al., 1999). DNA polymerase η
plays a role in error-free translesion DNA synthesis and
can bypass a cyclobutane pyrimidine dimer (CPD), a major UV-photoproduct, by introducing a correct nucleotide
opposite the lesion (Johnson et al., 1999). DNA polymerase η is deficient in cells from human patients with
Xeroderma pigmentosum variant (XP-V), a genetic disease associated with cancer susceptibility (Masutani et al.,
1999). Another yeast Rad30 homologue was discovered
in human cells and named DNA polymerase ι (Tissier et
al., 2000). However, unlike DNA polymerase η, DNA
polymerase ι is an error-prone enzyme. This is consistent
with the fact that deficiency in DNA polymerase η causes
XP-V, but deficiency in DNA polymerase ι does not.
DNA polymerase ξ has two polypeptide subunits encoded
by REV3 and REV7 in S. cerevisiae, respectively. Rev3p
has a conserved DNA polymerase B family motif (Fig. 1).
DNA polymerase ξ is involved in error-prone translesion
DNA synthesis, which causes damage-induced mutagenesis (Baynton et al., 1998; 1999). Rev1p, which is a member of the UmuC/DinB family, has deoxycitydyltransferase activity and can insert C opposite an abasic
site (Nelson et al., 1996). Four DNA polymerases - Rev1,
Polη, Polι, and Polκ - that belong to the UmuC/DinB
family have been isolated from human cells, but their
specific roles in vivo remain largely unknown.
Future directions and recent studies
DNA polymerases are important enzymes that play essential roles in DNA metabolism in all species from E. coli to
human. Five DNA-dependent DNA polymerases are
known in E. coli, nine in S. cerevisiae, and more than ten
in human cells. Due to ongoing genome projects, more
polymerases are being identified in mammalian species as
more DNA sequence information becomes available. The
recently identified polymerases include multiple transle-
Yasuo Kawasaki & Akio Sugino
sion DNA polymerases from E. coli (2), S. cerevisiae (3),
and human cells (4 or more). The translesion DNA polymerases appear to have specialized functions that recognize and repair different lesions in template DNA. Many
of the translesion DNA polymerases lack proofreading
activity and may cause damage-induced mutagenesis.
However, it is well established in bacteria that such errorprone DNA polymerases play an important role in enhancing survival in the presence of DNA-damaging
agents. One can easily imagine that such DNA polymerases might increase the (local) mutation rate, therefore
increasing genetic variability. However, the role(s) of
these translesion polymerases during meiosis are unknown.
Pre-meiotic DNA replication has not been studied extensively. As more is learned about DNA replication in
meiotic cells, it is anticipated that new and potentially
surprising results will emerge. For example, during meiosis, genomic regions can undergo drastic rearrangement
that involves homologous recombination and meiosisspecific double-strand breaks. These processes require
DNA synthesis and DNA polymerases. DNA polymerase
β, which was thought to function primarily in BER, is
now known to play a role in meiosis (Leem et al., 1994;
Plug et al., 1997).
DNA polymerases participate in many aspects of DNA
metabolism, which may be reflected in their specific subcellular or subnuclear localization. Eso1p was initially
isolated as a protein that is required for establishing sister
chromatid cohesion in S. pombe (Tanaka et al., 2000).
Recent results show that this protein has a DNA polymerase domain that is homologous to the UmuC/DinB
family. However, deletion of the DNA polymerase domain of Eso1p does not cause a defect in sister chromatid
cohesion, but does make mutant cells sensitive to UV
irradiation. This suggests that Eso1p might participate in
translesion DNA synthesis. It is interesting to consider the
possibility that DNA replication, post-replicational repair,
and sister chromatid cohesion may occur in a specific
subnuclear location, such as a “replication factory”.
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