REVIEWS
TIBS 25 – MARCH 2000
17 Lemon, B.J. and Peters, J.W. (1999) Binding of
exogenously added carbon monoxide at the active site
of the iron-only hydrogenase (CpI) from Clostridium
pasteurianum. Biochemistry 38, 12969–12973
18 Montet, Y. et al. (1997) Gas access to the active site
of Ni–Fe hydrogenases probed by X-ray crystallography
and molecular dynamics. Nat. Struct. Biol. 4, 523–526
19 Niu, S. et al. (1999) Theoretical characterization of the
reaction intermediates in a model of the nickel–iron
hydrogenase of Desulfovibrio gigas. J. Am. Chem. Soc.
121, 4000–4007
20 Gorwa, M.F. et al. (1996) Molecular characterization
and transcriptional analysis of the putative
hydrogenase gene of Clostridium acetobutylicum ATCC
824. J. Bacteriol. 178, 2668–2675
21 Malki, S. et al. (1995) Characterization of an operon
encoding an NADP-reducing hydrogenase in Desulfovibrio
fructosovorans. J. Bacteriol. 177, 2628–2636
22 Stokkermans, J. et al. (1989) hyd gamma, a gene from
Desulfovibrio vulgaris (Hildenborough) encodes a
polypeptide homologous to the periplasmic
hydrogenase. FEMS Microbiol. Lett. 49, 217–222
23 Voordouw, G. et al. (1985) Cloning of the gene
encoding the hydrogenase from Desulfovibrio vulgaris
(Hildenborough) and determination of the NH2-terminal
sequence. Eur. J. Biochem. 148. 509–514
24 Voordouw, G. et al. (1989) Organization of the genes
encoding [Fe] hydrogenase in Desulfovibrio vulgaris
subsp. oxamicus Monticello. J. Bacteriol. 171,
3881–3889
Eukaryotic DNA polymerases, a
growing family
Ulrich Hübscher, Heinz-Peter Nasheuer and
Juhani E. Syväoja
In eukaryotic cells, DNA polymerases are required to maintain the integrity
of the genome during processes, such as DNA replication, various DNA
repair events, translesion DNA synthesis, DNA recombination, and also in
regulatory events, such as cell cycle control and DNA damage checkpoint
function. In the last two years, the number of known DNA polymerases has
increased to at least nine (called a, b, g, d, e, z, h, u and i), and yeast
Saccharomyces cerevisiae contains REV1 deoxycytidyl transferase.
ANY LIVING CELL and organism is faced
with the tremendous task of keeping the
genome intact in order to develop in an
organized manner, function in a complex
environment, divide at the right time and
die when it is appropriate. To achieve
this, DNA synthesis is required to duplicate the genetic information prior to cell
division. DNA synthesis is also needed
during DNA repair processes, including
DNA recombination and bypassing lesions when the DNA has been damaged
(translesion DNA synthesis). DNA synthesis is performed by enzymes, called
DNA polymerases (DNA pol)1. Since the
U. Hübscher is at the Institute of Veterinary
Biochemistry, University of Zürich-Irchel,
Winterthurerstrasse 190, CH-8057 Zürich,
Switzerland; H-P. Nasheuer is at the Institute
of Molecular Biotechnology, Dept of
Biochemistry, Beutenbergstrasse 11,
D-07745 Jena, Germany; and J.E. Syväoja is
at the Biocenter Oulu and Dept of
Biochemistry, University of Oulu, FIN-90570
Oulu, Finland, and Dept of Biology, University
of Joensuu, FIN-80100 Joensuu, Finland.
Email: [email protected]
discovery of DNA pol a in eukaryotic
cells in 1957, the number of DNA pols
identified has grown. In the early 1970s,
DNA pol b and g were discovered leading
to the simple concept that DNA pol a is
the enzyme involved in DNA replication,
DNA pol b in DNA repair and DNA pol g in
mitochondrial DNA replication. However,
the discovery of DNA pol d and e in mammalian cells during the 1980s complicated this interpretation. It also suggested that a particular DNA pol might
have more than one functional task
in a cell, and that a particular DNA synthetic event can require more than one
DNA pol (reviewed in Ref. 2). Genetic
studies performed with budding yeast
Saccharomyces cerevisiae, for example,
showed that the three DNA pols a, d and
e share the task of replicating the cellular
genome, and that DNA repair events such
as base excision repair might require not
only DNA pol b but also DNA pol d or
DNA pol e, or both, especially in longpatch base excision repair. Because both
replication and repair are of primary
importance for cells and organisms, it
0968 – 0004/00/$ – See front matter © 2000, Elsevier Science Ltd. All rights reserved.
25 Santangelo, J.D. et al. (1995) Characterization and
expression of the hydrogenase-encoding gene from
Clostridium acetobutylicum P262. Microbiology 141,
171–180
26 Kraulis, P.J. (1991) MOLSCRIPT: a program to produce
both detailed and schematic plots of protein structures.
J. Appl. Crystallogr. 24, 946–950
27 Arnez, J.G. (1994) MINIMAGE: a program for plotting
electron-density maps. J. Appl. Crystallogr. 27,
649–653
28 Merritt, E.A. and Bacon, D.J. (1997) Raster3D
photorealistic molecular graphics. Methods Enzymol.
277, 505–524
29 Nicholls, A. (1992) GRASP: Graphical Representation
and Analysis of Surface Properties, Colombia University
appears that nature created safety mechanisms by employing various DNA pols
for similar functional tasks. Translesion
DNA synthesis, for example, requires at
least DNA pols z and h, the former probably being responsible for error-prone
translesion DNA synthesis and the latter
performing error-free DNA translesion
synthesis (reviewed in Ref. 3).
In many cases, DNA pols have complex
polypeptide structures because, in addition to the polymerizing subunit (that
often contains a proofreading 39→59 exonuclease), they comprise other functional subunits (Table 1). These functions
include other enzymatic activities (e.g.
DNA primase) or allow the DNA polymerase to interact with other proteins, involved in check-point function, cell cycle
control, DNA replication or DNA repair.
The replicative DNA pols d and e, for example, are chaperoned by the accessory
proteins replication factor C (RF-C), and
proliferating cell nuclear antigen (PCNA).
The interaction of these two accessory
proteins with the DNA pols permits a high
speed of DNA synthesis coupled with a
high accuracy4.
At the structural level, DNA pols appear to possess a universal DNA polymerase active site5. This is achieved by a
two-metal-ion-catalysed mechanism and
guarantees the incorporation of the correctly base-paired deoxyribonucleoside
triphosphate (according to the Watson–
Crick base pairing rule A–T and G–C)
onto a growing template. However, DNA
pols do differ in various aspects of their
structural architecture5, as a result of
the many possible interactions of DNA
pols with other proteins and enzymes.
Thus, the active site of the DNA pol is
very conserved in evolution, whereas
the structure of the surface of the molecules might differ considerably.
Functional roles of DNA pol a
DNA polymerase a–primase (DNA pol a–
prim) has an important role in DNA
replication (Fig. 1, reviewed in Ref. 6).
PII: S0968-0004(99)01523-6
143
REVIEWS
TIBS 25 – MARCH 2000
Table 1. The eukaryotic DNA polymerases: polypeptide compositions,
enzymatic activities and functionsa
Pol
Subunitsb
(M 3 103)
Enzymatic activity
Functional taskc
a
165
67
58
48
(167)
(79/86)
(58)
(48)
DNA pol
Replication: initiation; repair: DSBR; telomer
length regulation; cell cycle regulation
b
39
(68)
DNA pol, 59 phosphatase
Repair: BER, DSBR; meiosis
g
125 (143)
35
DNA pol, 39→59 exonuclease
Mitochondrial DNA replication
d
125 (125)
66 (55)
50 (40)
(22)d
DNA pol, 39→59 exonuclease
Replication: leading strand, lagging strand;
repair: MMR, DSBR, BER, NER; translesion
DNA synthesis; cell cycle regulation
e
261 (256)
59 (79)
(34)
(29)
DNA pol, 39→59 exonuclease
Replication: lagging strand; repair: DSBR,
BER, NER; cell cycle regulation
z
353 (173)
(29)
DNA pol
Error-prone translesion DNA synthesis
h
78
DNA pol (Rad30)
Error-free translesion DNA synthesis
u
198 ?
DNA pol, helicase?
Repair of interstrand crosslinks
i
?
?
DNA pol (Rad 30B)
Error-prone translesion DNA synthesis
(116)
Deoxycytidyl transferase
Abasic site synthesise
REV1
(70)
Primase
aFor
references, see text
Mammalian cells (yeast Saccharomyces cerevisiae)
DSBR, double-stranded break repair; BER, base excision repair; MMR, mismatch repair;
NER, nucleotide excision repair.
d
Schizosaccharomyces pombe has a fourth, possibly even a fifth subunit2
e
A new category of a nucleotide-polymerizing enzyme38
b
c
The DNA pol a–prim complex consists of
four subunits, with molecular masses of
165 kDa, 67–86 kDa, 58 kDa and 48 kDa.
The 48-kDa subunit is the only enzyme
that can start DNA synthesis de novo. It
also contains the catalytic centre of the
primase, although full activity is only
attained when the p48 and p58 subunits
come together to form the heterodimeric
primase. During initiation of the leading
strand, the DNA pol a–prim synthesizes a
short RNA primer at the replication
origin and the primer for the Okazaki
fragments on the lagging strand. Okazaki
fragments are the short DNA pieces at
the lagging strand of the replication fork
that are formed discontinuously because
all known DNA pols have a 59→39 directionality in synthesizing DNA. These
primers are then elongated to ~30–40
nucleotides by the intrinsic DNA pol
activity residing in the p165 subunit of
the heterotetrameric DNA pol a–prim.
The p67–86 (or B) subunit appears to
have no enzymatic functions but is
involved in the regulation of the initiation
reaction.
144
DNA pol a–prim also plays an important role in coordinating DNA replication, DNA repair and cell-cycle checkpoints6. It couples mitosis to the
completion of DNA replication and to the
repair of DNA damage, and is essential
for the arrest of yeast cells in G1 prior to
the start of S phase. These functions
might depend on the interaction of DNA
pol a–prim with other cellular factors involved in DNA metabolism and on its
regulation by checkpoint proteins, such
as yeast MEC1, a homologue of human
ATM (ataxia telangiectasia mutated)6.
Furthermore, cyclin-dependent kinases (CDK) phosphorylate and regulate DNA pol a–prim during the cell
cycle. Using the cell-free initiation of
SV40 DNA replication, it was shown that
cyclin-A-dependent kinases can inhibit
the initiation reaction, and that specific
amino acids of the p67–86 subunit are
essential for this regulation4,7. The loading of DNA pol a–prim into the chromatin might represent an additional
layer to control the initiation activity of
the enzyme complex8.
A direct role of DNA pol a–prim in
DNA repair and DNA recombination is
still under discussion2. Recent findings
suggested that all replicative DNA pols,
a, d and e, are needed for doublestranded break repair in yeast by homologous recombination with DNA pol
a–prim probably initiating DNA replication on the lagging strand9.
Functional roles of DNA pol b
Pol b is a single polypeptide of 39 kDa
comprising 335 amino acid residues (reviewed in Ref. 10). It consists of two domains connected by a proteasesensitive hinge region. The 8-kDa Nterminal domain carries dRpase (to remove the 59-deoxyribose phosphate)
and template-binding functions, whereas
the 31-kDa domain carries the DNA pol
activity (Table 1). The 3-D structure of
DNA pol b has been determined, and the
roles of critical amino acid residues in
the catalytic center have been studied5.
The 39OH primer terminus is perfectly
positioned in the active site to conduct a
nucleophilic attack on the a-phosphorus
of
the
incoming,
base-pairing
deoxyribonucleoside-59triphosphate. This
mechanism gurantees the proper chain
elongation according to the base pair
rule A–T and G–C. The 3-D structure of
DNA pol b and mutagenesis studies have
revealed that three aspartic acid
residues in the active site are involved in
dNMP transfer – like corresponding aspartic acid residues in the active site of
Escherichia coli DNA pol I Klenow fragment. However, the template is probably
bound to DNA pol b in a manner that is
different from other DNA pols.
DNA pol b has its role in DNA repair2.
There are two overall biochemical pathways of base excision repair in mammalian cells: short-patch (or singlenucleotide-replacement) base excision
repair and long-patch (or severalnucleotide-replacement) base excision
repair10. Several lines of evidence have
implicated DNA pol b in mammalian
short-patch base excision repair. In this
pathway, incision on the 59 side of the
apurinic or the apyrimidinic site is followed by removal of the sugar phosphate by the dRpase activity of DNA pol
b creating a one nucleotide gap flanked
by 39-OH and 59-phosphate. DNA pol d
or e are probably involved in PCNAdependent, long-patch base excision
repair with gaps of 2–13 nucleotides11,12.
In this pathway, the flap endonuclease 1
(Fen 1) is needed to cleave a reaction intermediate generated by template
strand displacement during gap filling.
REVIEWS
TIBS 25 – MARCH 2000
A variation of the long-patch base
excision repair pathway with gap
lengths of 2–6 nucleotides has also been
characterized in mammalian cells. This
Fen-1- and PCNA-dependent pathway
can be reconstituted with DNA pol d
and DNA pol e, although recent experiments also suggested that DNA pol b
can act in this pathway in vivo13, as
suggested by the reduced repair activity in DNA pol-b-deficient cells, and in
the presence of DNA-pol-b-neutralizing
antibody.
It appears that DNA pol b acts in
meiosis and double-stranded break repair. First, DNA pol b has also been implicated in the meiotic events associated with synapsis and recombination14.
Second, a 67-kDa homologue of mammalian DNA pol b, encoded by the nonessential POL4 gene (Table 1) of S. cerevisiae, has recently been implicated in
double-stranded break repair, probably
utilizing a nonhomologous-end-joining
mechanism15.
Functional roles of DNA pol d
Although DNA pol d was discovered in
1976, it took more than a decade for this
enzyme to earn its place as a DNA polymerase1. The reason being that DNA pol
d had to be considered in the context of
the two factors PCNA and RF-C. These
protein complexes build the moving
platform for DNA pol d (Ref. 16) and
therefore, provide an important framework for the dynamic properties of the
DNA polymerase. These properties include the recruitment of DNA pol d and a
primer terminus occupied by DNA pol a
(the DNA polymerase switch4; see Fig. 1),
the increase of DNA pol processivity, the
prevention of non-productive binding to
single-stranded DNA, the release of DNA
pol d after DNA synthesis and the bridging to other proteins involved in DNA
metabolic events. DNA pol d exists as a
heterodimeric enzyme with subunits of
120 and 50 kDa (Ref. 2), although recent
work has suggested a more complex
polypeptide structure of DNA pol d (see
Table 1; reviewed in Ref. 2).
DNA pol d possesses a wide range of
functions – some of which have already
been discussed2 – and is needed in DNA
replication and in several DNA repair
events (Table 1). According to models
proposed by Burgers2, DNA pol d fullfils
two roles in DNA replication, namely at
the leading strand and at the lagging
strand, where (probably in cooperation
with DNA pol e) it elongates and
maturates Okazaki-fragments (Fig. 1).
Nucleotide excision repair can be carried
Pre-initiation
Initiation and post-initiation
ORI
ORI
Binding of
replication
origins
(1)
ORI
ORC/Tag
leading-strand
DNA pol δ/ε
ORI
Loading of
replication
factors
(2)
Maturation of Okazaki
fragments, termination
of DNA replication, etc.
(8)
Okazaki fragments:
DNA pol δ, or ε,
or both?
CDC6/Tag:
MCMs,
RP-A, etc.
ORI
Polymerase switch on
the lagging strand
(7)
DNA pol δ or
DNA pol ε
Destabilization
of dsDNA
(3)
leading-strand
DNA pol δ/ε
MCM4-6-7?/
Tag
ORI
lagging-strand
DNA pol α–prim
topo I
Unwinding
of dsDNA
(4)
Leading- and laggingstrand synthesis
(6)
PCNA, RF-C,
DNA pol δ, DNA pol ε
ORI
helicase
DNA pol α−prim
Initiation of leadingstrand synthesis
(5)
Tag
DNA pol α−prim
RF-C
DNA pol δ
RP-A
topo I
PCNA
DNA pol ε
RNA−DNA
primer
Ti BS
Figure 1
DNA polymerases in eukaroytic DNA replication. Detailed analysis of simian virus 40 (SV40)
DNA replication has led to a model for DNA replication of eukaryotic chromosomal DNA. In
the initial step, (1) T antigen (Tag) functions as a viral origin recognition complex (vORC)
comparable to the cellular ORC found in all eukaryotic cells. (2) Tag recruits the singlestranded DNA-binding factor replication protein-A (RP-A) and DNA topoisomerase I (topo I) to
(3) destabilize and (4) unwind double-stranded (ds) origin DNA. (5) Next, Tag loads the DNA
polymerase a–primase (DNA pol a–prim) onto the DNA. The primase enzyme of DNA pol a–prim
initiates initial leading-strand replication. Thus, these activities of Tag resemble those of
several cellular proteins, such as CDC6 (CDC stands for cell division cycle) and CDC45,
which load replication factors onto the chromatin (Refs 8,26 and references therein). The
DNA pol a–prim synthesizes the primer RNA molecules that are elongated by the intrinsic
DNA polymerase activity. (6) These RNA–DNA molecules are then recognized by the replication factor C (RF-C), which loads proliferating cell nuclear antigen (PCNA) onto the
. The latter tethers DNA pol d and probably DNA pol e onto the primer terminus. The leading
strand is then replicated by DNA pol d. (7) The Okazaki fragments on the
lagging-strand are initiated by DNA pol a–prim and synthesized by RF-C, PCNA and DNA
pol d and possibly also DNA pol e. (8) The maturation of the Okazaki fragments requires a
whole set of additional proteins and DNA pol d or e (for reviews, see Refs 2 and 4).
out by DNA pol d or DNA pol e holoenzyme in vitro (comprising DNA pol d or e,
PCNA and RF-C)17. Mismatch repair relies
on DNA pol d for DNA synthesis. Following
the initial notion that PCNA is required for
this repair event18, it has been found that
DNA pol d is required for efficient in vitro
repair of mismatched DNA19. Genetic studies in yeast Saccharomyces cerevisiae suggested that a set of repair pathways falls
within the so-called RAD6 epistasis group
and deals with the bypass of DNA damage
145
REVIEWS
Functional roles of DNA pol e
The genes encoding the 256-kDa catalytic and 79-kDa subunit of S. cerevisiae
DNA pol e are essential for growth22, as is
the catalytic subunit of S. pombe DNA pol e
(Ref. 23). Requirement of the catalytic
subunit has been taken to reflect the
need for DNA polymerase activity of DNA
pol e in replication in yeast cells.
However, the precise role of DNA pol e
both in yeast and in mammalian cells remains to be determined. The three DNA
pols a, d and e can be UV-crosslinked to
nascent cellular DNA (Ref. 24), and neutralizing antibodies against DNA pol e
inhibited replicative DNA synthesis both
in nuclei of growing cells and in isolated
nuclei25, suggesting that DNA pol e is
involved in DNA synthesis of replicating
chromosomes in mammalian cells.
Crosslinking studies in S. cerevisiae have
also indicated that DNA pol e resides at
or close to replication forks in S-phase
cells26. Recently, the question concerning
the role of DNA pol e in replication has
become even more intriguing because
the catalytic DNA pol domain within the
256-kDa polypeptide is not essential in
budding yeast27. In addition to DNA replication and viability, cells that lack the
catalytic DNA pol domain are also proficient in DNA repair and recombination.
Although genes encoding both the 256kDa and the 79-kDa subunit are required
for S. cerevisiae, the DNA pol e catalytic
domain is not. This raises the question of
the essential role of DNA pol e and where
in the protein this functionality resides. It
is the C-terminal portion of the 256-kDa
catalytic subunit that is both necessary
and sufficient for all of the essential functions of S. cerevisiae DNA pol e (Fig. 2 and
Ref. 27). Further mapping has highlighted
the importance of a cysteine-rich stretch
with two zinc fingers in the extreme C terminus28. The C-terminal region has been
proposed to act as a sensor of DNA replication that coordinates the transcriptional and cell cycle responses to replication blocks or DNA damage during
146
Zn
-fi
ng
er
pol ε
N
exter
o m
po
l
Essential for
viability in yeast
e
pol δ
pol α
ex
olik
that forms blocks during DNA replication20. Besides the pivotal roles of DNA
pol z, DNA pol h and Rev 1 in this repair
translesion synthesis events (see below),
DNA pol d also appears to function in
translesion DNA synthesis20. Different
temperature-sensitive mutants within the
large 125-kDa subunit of DNA pol d have
been identified in Schizosaccromyces
pombe that exhibit a typical ‘cell division
cycle’ terminal phenotype, suggesting
that DNA pol d is also involved in cell
cycle control21.
TIBS 25 – MARCH 2000
pol ζ
200 aa
Ti BS
Figure 2
The domain structure of human pol e
compared with the structure of the related human pols a, d and z. The N-terminal (N-term), proofreading exonuclease
(exo), polymerase (pol) domains and the
putative C4 zinc fingers are indicated in
green, dark blue, red and yellow, respectively. Although DNA pol a and DNA pol z
do not exhibit any proofreading exonuclease activity, regions of similarity to the
exonuclease domain can be detected in
their primary structure (hatched). The putative zinc fingers are less conserved in
DNA pol a than in the other DNA pols.
The region of DNA pol e that is essential
for cell viability in Saccharomyces cerevisiae has been highlighted in light blue.
S phase in S. cerevisiae29. However, DNA
pol e does not seem to be required for
S-phase checkpoints in S. pombe23.
Besides DNA replication, it is likely
that DNA pol e plays a role in nucleotide
excision repair, given that DNA pol e
or DNA pol d are required in the DNA
synthesis process of reconstituted nucleotide excision repair17. Using soluble
yeast repair extracts from mutant budding yeast strains, Wang et al.30 found
that DNA pol e rather than DNA pol d is
required for base excision repair. DNA
pol e and DNA pol d are also redundant in
mammalian long-patch base excision
repair (see above).
DNA pols d, z, h, i and Rev 1 in translesion
DNA synthesis
Replicative DNA pols stop when they
encounter a DNA lesion31. On the leading
strand the replication fork is blocked,
whereas lagging strand synthesis is only
partially interrupted. Due to its discontinuous mode, DNA synthesis will continue after initiation of a new Okazaki
fragment. Therefore, additional processes are required to bypass or to repair lesions. Recently, novel insights
were gained into this poorly understood
mechanism. In eukaryotes, besides DNA
pol d, at least DNA pol z and DNA pol h
bypass DNA lesions. The latter two DNA
pols are non-essential for DNA replication but are conserved from yeast
to mammals32–36. DNA pol z and h are
involved in two independent pathways:
error-prone and error-free bypass of
thymine–thymine cyclobutane (TT)
dimers. In yeast, the Rev3 and Rev7
genes are required for damage-induced
mutagenesis and code for the DNA pol z
complex, with Rev3p carrying the catalytic function37. DNA pol z can synthesize DNA past a lesion with low efficiency and frequent errors. In contrast,
DNA pol h, the product of the yeast
Rad30 gene, efficiently bypasses TT dimers with high fidelity33. DNA pol h is
homologous to UmuC and DinB, which
are required for translesion synthesis in
E. coli33–35, and to Rev1, which is involved in translesion synthesis in yeast.
Rev1p contains deoxycytidyl transferase
activity and inserts C opposite an abasic
site38. Moreover, human pol h complements the cancer-prone genetic disease Xeroderma pigmentosum variant
(XP-V), as the DNA pol h gene is mutated
in XP-V cells34,39. Finally, the recently
identified Rad30B homolog in human
cells40 is called DNA pol i (R. Woodgate,
pers. commun.).
The precise roles of these novel pols
are far from being understood in detail
but these new findings suggest that
Rev1, DNA pol z , h and i are involved in
translesion synthesis and can probably
bypass different kinds of DNA lesions.
Conclusion and outlook
The DNA pol family in eukaryotic
cells is still growing (reviewed in Ref.
36). Recent cloning and chromosomal
mapping brought to light a ninth DNA
pol in human cells, called DNA pol u
(Ref. 41) The findings that DNA pol h
and DNA pol z have bacterial homologues, which have been identified recently as DNA pols [Din B as pol IV
(Ref. 42) and Umu D92C as pol V
(Ref. 43)], shed new light on the mechanisms of DNA synthesis in Escherichia
coli and their conservation from
prokaryotes to eukaryotes. The novel
translesion DNA pols are also enzymes
of evolutionary change44. Furthermore,
new experimental data have led to the
picture that eukaryotic DNA pols have
cross-functionalities in DNA replication,
various DNA repair events and DNA recombination. In addition, they are also
involved in control mechanisms governing cell cycle and checkpoints. We can
look forward to an exciting time in
COMPUTER CORNER
TIBS 25 – MARCH 2000
DNA pol research, with new DNA pol
discoveries bound to occur in the near
future.
Acknowledgements
We thank H. Pospiech, R. Smith, F.
Grosse and S. Hasan for critical reading
of the manuscript and for help with
the artwork. U.H. has been supported
by the Swiss National Science
Foundation and the Kanton of Zürich.
H.P.N. is supported by the Deutsche
Forschungsgemeinschaft
and
the
Boehringer Ingelheim Fund. J.E.S. is
supported by the Academy of Finland.
H.P.N. and U.H. are participating in the
Training Mobility and Research grant
ERBMRXCT CT970125. We apologize to
those authors whose work could only
be cited indirectly in recent reviews
owing to editorial limitations.
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NPS@: Network Protein Sequence
Analysis
A large number of sequences are being
generated by the various genome sequencing projects. One of the major
challenges in the biocomputing field is
to derive valuable information from
these protein sequences. The first prerequisite in this process is to access
up-to-date sequence and structure
databanks (e.g. EMBL, GenBank, SWISSPROT, Protein Data Bank; for a catalogue,
see Ref. 1) maintained by several biocomputing centres, such as NCBI, EBI,
EMBL, SIB and INFOBIOGEN. Ideally, sequences are analysed using a maximal
number of methods on a minimal number of different Web sites. To achieve
this, we developed a Web server called
NPS@ (Network Protein Sequence
Analysis, http://pbil.ibcp.fr/NPSA) that
became available in 1998. NPS@ is the
protein sequence analysis Web server
of the Pôle BioInformatique Lyonnais,
a group of biocomputing teams
(http://pbil.univ-lyon1.fr), and provides
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Kesti,T. et al. (1999) DNA polymerase e catalytic domains
are dispensible for DNA replication, DNA repair and cell
viability, Mol. Cell 3, 679–685
Dua, R. et al. (1999) Analysis of the essential functions
of the C-terminal protein/protein interaction domain of
Saccharomyces cerevisiae pol e and its unexpected
activity to support growth in the absence of the DNA
polymerase domain. J. Biol. Chem. 274, 22283–22288
Navas, T. et al. (1995) DNA polymerase e links the DNA
replication machinery to the S phase check point. Cell
80, 29–39
Wang, Z. et al. (1993) DNA repair synthesis during base
excision repair in vitro is catalysed by DNA polymerase e
and is influenced by DNA polymerases a and d in
Saccharomyces cerevisiae. Mol. Cell. Biol. 13, 1051–1058
Friedberg, E.C. and Gerlach, V.L. (1999) Novel DNA
polymerases offer clues to the molecular basis of
mutagenesis. Cell 98, 413–416
Gibbs, P.E. et al. (1998) A human homolog of the
Saccharomyces cerevisiae REV3 gene which encodes the
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the user with many of the most commonly used tools for protein sequence
analysis.
The general flowchart describing relationships between biological sequence
data and biocomputing methods available on the NPS@ web server is given in
Fig. 1. The main feature of NPS@ is the
interconnection of all methods gathered
within a simple and user-friendly Web
interface. Thus, it provides an easy
way for protein sequence analysis and
avoids the rather tedious cut-and-paste
operations between sites. Therefore, on
the same server the user can (1) search
for homologous protein sequences; (2)
constitute a subset of matching protein
sequences; (3) perform multiple alignments; (4) make secondary structure
predictions, then generate a consensus
PII: S0968-0004(99)01540-6
147