Eukaryotic DNA Polymerase d
P M J Burgers, Washington University School of Medicine, St. Louis, MO, USA
ã 2013 Elsevier Inc. All rights reserved.
This article is a revision of the previous edition article by Antero G. So and Kathleen M. Downey, volume 1, pp. 713–715, ã 2004, Elsevier Inc.
Structure
Catalytic Subunit
Pol d is a multisubunit complex, consisting of a catalytic sub-
unit, and, depending on the organism, two or three accessory
subunits (Table 1). The catalytic subunit is essential for yeast
cell growth and belongs to the B-family of DNA polymerases
found in all three kingdoms of life.
The simplest B-family DNA polymerase is the replica-
tive DNA polymerase of bacteriophage RB69. Its structure
shows three distinct domains: a large N-terminal domain,
the 30 -exonuclease domain, and the polymerase domain. The
polymerase domain shows a structure that is typical for DNA
polymerases with the template primer junction and the dNTP
binding into a broad cleft that closes up into a closed complex
prior to the chemical polymerization step. The single-stranded
template DNA enters the active site from a cleft situated
between the N-terminal domain and the exonuclease domain,
whereas the double-stranded DNA exits the active toward the
bottom in Figure 1(a). The active site of the polymerase
domain is precisely defined to promote favorable binding of
that incoming dNTP that will form a proper Watson–Crick
base pair with the next template nucleotide to be replicated,
whereas non-Watson–Crick base pairs show a poor fit and are,
therefore, disfavored. These geometric considerations, together
with the favorable energetics obtained from Watson–Crick
base pairing, result in a high fidelity of replication. Yeast Pol
d has an error rate of about 104–105, that is, there is one
misincorporation event for every 104–105 nucleotides (nt)
replicated. The fidelity of DNA replication by Pol d is enhanced
about 10-fold through proofreading by its 30 –50 -exonuclease
activity.
The 30 -exonuclease domain of Pol d belongs to the same class
as those identified in other proofreading DNA polymerases.
Two divalent metals in the exonuclease domain coordinate
with aspartates/glutamates to orient a water molecule as a
metal-hydroxide ion for attack of the internucleotide phospho-
diester bond to be cleaved. The active site of the 30 -exonuclease
domain is 35 Å distant from the active site of the polymerase
domain. Misincorporation by the polymerase results in stalling
of further DNA synthesis because of a poor geometric fit of the
mismatched template–primer junction in the active site of
the polymerase domain. This stalling promotes dissociation
of the primer terminus from the polymerase domain and
binding in the exonuclease domain. Although it is yet unclear
how this translocation from the polymerase to the exonuclease
domain occurs, it is an important step in maintaining replica-
tion fidelity, and it is just as important that this translocation is
reversible so that polymerization can resume after hydrolysis
of the misincorporated nucleotide. The relative orientation of
the polymerase and exonuclease domains in B-family DNA
polymerases is completely different from that in the A-family
enzymes; yet both types of enzymes evolved a pathway of facile
shuttling of the primer terminus between the two domains.
The overall structure of the N-terminal domain is also largely
conserved between various B-class DNA polymerases. It forms
a three-lobed structure, with one lobe showing similarity
with single-stranded DNA-binding motifs and a second lobe
showing similarity with ribonucleic acid (RNA)-recognition
motifs. Given that all crystal structures of polymerase–DNA
complexes show the single-stranded template DNA extruding
toward a cleft between the N-terminal domain and the exonu-
clease domain, a likely function of the N-terminal domain is to
bind the downstream single-stranded template DNA. In addi-
tion, Pol d is involved in several pathways that require gap-
filling reactions including those that occur during Okazaki
fragment maturation on the lagging strand of the replication
fork. Therefore, the N-terminal domain may recognize the
downstream 50 -single-/double-stranded DNA junction during
gap filling.
In addition to these three conserved domains shared with
all B-family DNA polymerases, the cellular B-family enzymes
uniquely contain a highly conserved C-terminal domain. This
domain shows a typical structural arrangement of conserved
cysteine residues that chelate divalent metals. In the case of Pol
a, two Zn ions are found at these sites, and it is these motifs
that mediate interactions with the other subunits of the com-
plex. In addition to the structural functions ascribed to this
C-terminal domain, it may also play a specialized function in
mediating damage-induced mutagenesis in the cell as muta-
tions in the domain are specifically defective for this process.
Associated Subunits
The second subunit of Pol d shows considerable structural
homology with the second subunits of Pol a and Pol e. Given
the high conservation of the metal-binding domains of the
C-termini of the catalytic subunits, it is likely that subunit–
subunit interactions between each of the three catalytic sub-
units with their respective second subunits are also conserved.
The proposed subunit–subunit interface between yeast Pol3
and Pol31 in Figure 1(b) is based on the crystal structure of
the homologous subunit interactions between the catalytic and
second subunit of Pol a. A possible function for an OB-fold
domain (oligonucleotide/oligosaccharide binding) in Pol31,
beyond its involvement in subunit interactions, remains to be
established. OB-fold domains are often active in binding
single-stranded DNA.
The third subunit of Pol d consists of a 15 kDa N-terminal
domain that mediates interactions with the second subunit,
followed by a largely unstructured domain of 20–45 kDa, and
a proliferating cell nuclear antigen (PCNA)-binding domain at
the extreme C-terminus of the subunit. In addition, interac-
tions with Pol a and with Rev1, a subunit of the cellular
245
246 Molecular Biology | Eukaryotic DNA Polymerase d

mutagenesis complex, have been identified. The presence of The PCNA-binding domain at the C-terminus of the third
the middle domain is responsible for the highly elongated subunit of Pol d is responsible for DNA-independent interac-
structure of this subunit, and consequently of the entire Pol d tions with PCNA. When both PCNA and Pol d are assembled
complex. Saccharomyces cerevisiae Pol32 is dispensable for onto DNA, additional PCNA-interaction domain(s) become
growth, but the deletion shows defects in DNA replication, active in promoting processivity. The precise domain(s) in
mutagenesis, and in break-induced DNA replication. By con- Pol d responsible for these DNA-dependent interactions with
trast, the S. pombe Cdc27 gene is essential. PCNA still remain to be mapped. However, PCNA-dependent
A small fourth subunit of Pol d has been identified processive DNA replication requires at a minimum both the
in human and S. pombe, but appears to be lacking from catalytic and second subunit of Pol d.
S. cerevisiae Pol d. This subunit enhances the stability of the
complex. It may also play a regulatory role during damage
response in the cell as this subunit is specifically degraded Functions of Pol d
after DNA damage.
The two catalytic functions of Pol d, that is, polymerization and
30 -exonuclease, support high-fidelity DNA replication, a neces-
PCNA as Processivity Factor sary function for genome maintenance. In addition, these two
catalytic functions have been adapted for specialized functions
Pol d has a very low processivity of DNA synthesis, which of Pol d, in particular those involving gap-filling reactions.
would make it an unsuitable enzyme for replicating long tem- During gap filling, when the downstream 50 ds/ss junction
plates with high efficiency. However, its interaction with PCNA is reached, Pol d can invade the double-stranded region
transforms Pol d into a highly processive enzyme, capable of and, thus, carry out strand-displacement synthesis. Remark-
replicating thousands of nucleotides without dissociating ably, however, strand-displacement synthesis is limited to
from the DNA. PCNA is a donut-like homotrimer that encir- only a few nucleotides producing a very short 50 -flap that,
cles dsDNA and binds proteins, thereby locking them onto depending of the pathway involved, is cleaved by an appropri-
the DNA. In addition to Pol d, almost a hundred other ate 50 -exonuclease. In the absence of this processing by the
PCNA-interacting proteins have been identified that function 50 -exonuclease, Pol d switches the primer terminus to its
in DNA metabolism and cell-cycle control. 30 -exonuclease domain that degrades the extended terminus
back to the nick position. The recurring process of extension
Table 1 Subunit structure of Pol d and degradation, called idling, allows Pol d to dynamically
Genes and subunit sizes Function
maintain the primer terminus either at the nick position, to
promote ligation of the nick, or at a 50 flap position, for degrada-
S. cerevisiae S. pombe Human tion by a 5’-exonuclease (Figure 2(a)). Idling prevents the gener-
ation of long flaps that can be a source for genomic instability.
POL3 125 Pol3 124 POLD1 124 Polymerase, 30 -exonuclease
POL31 55 Cdc1 51 POLD2 51 Required for PCNA-
dependent replication
DNA Replication
POL32 40 Cdc27 42 POLD3 66 PCNA binding
- Cdm1 19 POLD4 12 Complex stability
Recent studies have established that leading-strand DNA repli-
Gene designations and subunit sizes in kDa are given. cation is carried out by Pol e, while Pol d is the lagging-strand

N-terminal 3⬘-Exonuclease p50 (Pol31)

domain domain

Pol1
C-terminal
domain

Polymerase
domain
p66 N-terminal
Template Primer domain (Pol32N)
(a) (b)

Figure 1 Structural components of Pol d. All structures were rendered using Chimera, from coordinates deposited in the PDB database. (a) The
S. cerevisiae Pol3 polypetide, aa 95–985, in a complex with DNA (3IAY). The N-terminal, exonuclease and polymerase domains are indicated.
The C-terminal domain of Pol3 is missing from this structure. (b) The C-terminal domain (3FLO) of S. cerevisiae Pol 1 (catalytic subunit of Pol a),
which is highly homologous to that of Pol3, is docked onto the structure (3EOJ) of the human hetrodimeric complex of p50 with p66 (N-terminal
domain). The proposed connectivity between the N-terminal and C-terminal domains of Pol3 is indicated with a double gray arrow.
 Molecular Biology | Eukaryotic DNA Polymerase d 247

dNTP switch to a mutagenic form of DNA replication that involves

5⬘ 5⬘-Pol low-fidelity DNA polymerases that replicate across the dam-
Idling
3⬘ aged template, a process called translesion synthesis (TLS).
3⬘-Exo
The activity of the low-fidelity TLS Pol z forms the basis for
(a) dNMP
damage-induced mutagenesis in eukaryotic cells. Mutagenesis
is a highly controlled process that involves the participation of
Lagging several other factors. Genetic studies have shown that Pol d is
strand RNA
one of the factors essential for damage-induced mutagenesis.
replication 5⬘
3⬘ This is rather unexpected because Pol d is a high-fidelity
enzyme that would be very inefficient in TLS. Possibly, Pol d
has a role in organizing the mutagenic complex during TLS.
Okazaki
fragment
maturation 5⬘
3⬘ DNA Recombination

5⬘ The repair of double-stranded breaks in the cell by homolo-

Homologous 3⬘
DNA
recombination 3⬘
5⬘
Base
Damage
excision 5⬘
repair 3⬘
Nucleotide
excision 5⬘
repair 3⬘
Damage-
+TLS Pols
induced 5⬘
mutagenesis 3⬘
(b)
Figure 2 Activities and pathways of Pol d. (a) Idling is the result of
recurring cycles of polymerization and exonuclease activity. (b) Pathway
involvement for Pol d. See text for details. TLS, translesion synthesis.
DNA replicase. However, in the case of dysfunction of the
leading strand replicase Pol e, Pol d can, in addition, perform
leading-strand DNA replication. The ability of Pol d to function
on both strands may be important in some DNA damage-
response pathways that operate at damaged or unusual DNA
replication forks. Each Okazaki fragment on the lagging strand
is initiated by the primase activity of Pol a–Primase. After a
switch to Pol d, the enzyme can correct replication errors that
were made by Pol a during initiation. Pol d elongates Okazaki
fragments, which are 150 nt in length, and also matures these
fragments. The predominant pathway for maturation proceeds
in cooperation with the 50 -exonuclease FEN1 and DNA ligase I.
It is during maturation that the strand-displacement activity of
Pol d becomes important. By carrying out strand displacement
synthesis, Pol d dynamically generates the substrate for the
FEN1 50 -flap exonuclease ensuring degradation of the RNA
primer (Figure 2(b)). Once the RNA is degraded, DNA ligase
I can seal the nick. All of these reactions are coordinated by
interactions of each of these factors with PCNA.
When replication forks stall because of the presence of
template DNA damage, several mechanisms are available
to deal with this damage. One of these mechanisms is to
gous recombination is the most accurate repair mechanism as
it uses the information on the homologous chromosome to
repair the information lost at the break site. Homologous
recombination is initiated by strand-specific resection from
the double-stranded break in order to generate long 30 -single-
stranded tails. These 30 -tails invade the intact homologous
chromosome using standard leading to the formation of a
D-loop. DNA synthesis using the 30 -terminated strand of
the D-loop as a primer is an essential step in homologous
DNA recombination as it restores the information lost at the
initial break site. The strand-displacement synthesis in this
step is carried out by Pol d and its interaction with PCNA is
essential in the initiation of this process. Resolution of recom-
bination intermediates restores the proper genetic identity of
the broken chromosome.
Double-stranded breaks can also be generated by collapse
of stalled replication forks or by gradual degradation of the
telomeric ends of chromosomes. Such breaks have only one
broken end and do not undergo classical recombination.
Rather, after strand invasion, they establish a replication fork
that proceeds to the end of the invaded homologous chromo-
some, hence break-induced replication (BIR). During the initial
stages of BIR, replication is carried out by Pol d. Remarkably,
in the absence of the nonessential Pol32 subunit of Pol d, BIR is
largely defective. Only at later stages of BIR is a more standard
replication fork generated that also contains Pol e.
DNA Repair
Among the various DNA repair pathways, both base exci-
sion repair (BER) and nucleotide excision repair (NER)
involve a DNA synthesis step. Two distinct pathways exist for
removal of damaged nucleotides in BER. After 50 -incision
of the damaged strand, repair can proceed either through a
Pol b-dependent pathway or through a Pol d-dependent path-
way. The latter pathway is also called long-patch or PCNA-
dependent BER. The damage is removed by PCNA-dependent
strand-displacement synthesis by Pol d coordinate with PCNA-
promoted excision of the damaged strand by FEN1. This path-
way is comparable to that of RNA degradation during Okazaki
fragment maturation. In contrast to BER, it is less clear which
DNA polymerases carry out the required synthetic step during
NER. NER proceeds by excision of a 30-nt section of
248 Molecular Biology | Eukaryotic DNA Polymerase d
single-stranded DNA surrounding the DNA damage leaving a
gapped DNA intermediate. Genetic studies in yeast have impli-
cated both Pol d and Pol e as enzymes capable of carrying out
gap-filling synthesis. However, there is evidence that in human
cell, the low-fidelity Pol k may also serve a role in gap filling.
See also: Molecular Biology: DNA Mismatch Repair in Disease and
Aging; DNA Polymerase I, Bacterial; DNA Replication: Initiation in
Bacteria; Repair of G–T Mismatches by Escherichia coli Vsr and
Eukaryotic DNA Glycosylases.
Further Reading
Burgers PM (2009) Polymerase dynamics at the eukaryotic DNA replication fork.
Journal of Biological Chemistry 284: 4041–4045.
Hitomi K, Iwai S, and Tainer JA (2007) The intricate structural chemistry of base
excision repair machinery: Implications for DNA damage recognition, removal, and
repair. DNA Repair 6: 410–428.
Jain R, Hammel M, Johnson RE, et al. (2009) Structural insights into yeast DNA
polymerase delta by small angle X-ray scattering. Journal of Molecular Biology
394: 377–382.
Johansson E and Macneill SA (2010) The eukaryotic replicative DNA polymerases take
shape. Trends in Biochemical Sciences 35: 339–347.
Kunkel TA and Burgers PM (2008) Dividing the workload at a eukaryotic replication
fork. Trends in Cell Biology 18: 521–527.
Lydeard JR, Jain S, Yamaguchi M, and Haber JE (2007) Break-induced replication and
telomerase-independent telomere maintenance require Pol32. Nature
448: 820–823.
Relevant Website
http://www.cgl.ucsf.edu – UCSF Chimera.