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J. Biol. Chem., Vol. 278, Issue 36, 34685-34690, September 5, 2003
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From the
Laboratories of Molecular Genetics and
Structural Biology, NIEHS, National Institutes of Health, Department of Health
and Human Services, Research Triangle Park, North Carolina 27709 and
¶Centro de Biología Molecular Severo Ochoa
(CSIC-UAM), Universidad Autónoma, 28049 Madrid, Spain
Received for publication, June 2, 2003 , and in revised form, June 23, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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(Pol ) is a member of the Pol X family
having properties in common with several other mammalian DNA polymerases. To
obtain clues to possible functions in vivo, we have determined the
fidelity of DNA synthesis by human Pol . The results indicate that the
average single-base deletion error rate of Pol is higher than those
of other mammalian polymerases. In fact, unlike other DNA polymerases, Pol
generates single-base deletions at average rates that substantially
exceed base substitution rates. Moreover, the sequence specificity for
single-base deletions made by Pol is different from that of other DNA
polymerases and reveals that Pol readily uses template-primers with
limited base pair homology at the primer terminus. This ability, together with
an ability to fill short gaps in DNA at low dNTP concentrations, is consistent
with a role for mammalian Pol in non-homologous end-joining. This may
include non-homologous end-joining of strand breaks resulting from DNA damage,
because Pol has intrinsic 5',2'-deoxyribose-5-phosphate
lyase activity. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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is a
member of family X that was discovered only 3 years ago
(4–6).
Sequence comparisons have shown that it has 33% amino acid identity and shares
the same domain organization with the best studied member of family X, DNA Pol
, an enzyme crucial in base excision repair
(7). Members of the family X
also include terminal transferase (TdT)
(8), Pol µ
(9), and Pol 
(10). Despite sharing a
significant sequence similarity, the small, monomeric enzymes of this family
possess remarkably different properties. For example, TdT is a
template-independent polymerase involved in antigen receptor diversification
during V(D)J recombination
(11,
12). In contrast Pol is
a moderately faithful, template-dependent enzyme
(13,
14) that, in addition to its
polymerase activity, possesses a 5', 2'-deoxyribose-5-phosphate
(dRP) lyase activity (15,
16). Finally Pol µ, likely
involved in non-homologous endjoining (NHEJ)
(17), is a largely
template-dependent polymerase that is also endowed with some
template-independent polymerization activity
(9). Unlike Pol , Pol
µ is highly error-prone (Refs.
9 and
18).2
Early studies concluded that Pol is predominantly expressed in
testis in stages of spermatogenesis coincident with meiotic recombination
(4). The generation of
knock-out mice has, as yet, not confirmed the involvement of Pol in
this or any other process
(19). Although the biological
role of Pol is currently unknown, insights into its possible cellular
functions come from studies of its biochemical properties. Unlike TdT, Pol
is a template-dependent DNA polymerase
(20,
21). Like most other members
of family X, Pol lacks intrinsic 3'
5' exonuclease
activity (5,
20) and, therefore, cannot
proofread any errors it generates. Like Pol , Pol is
distributive on an "open" template-primer (e.g. a
template hybridized with a single primer), but it is processive when filling
short gaps (e.g. 1–5 nucleotides) in DNA if the 5' end of
the gap contains a phosphate group
(20). Moreover, Pol
has an intrinsic dRP lyase activity, and it can replace Pol to conduct
single-nucleotide base excision repair in vitro
(22). These properties make
Pol a suitable candidate to perform some form of DNA repair in
vivo. Interestingly, Pol has high affinity for dNTPs
(20), which suggests its
possible involvement in DNA transactions occurring under low concentrations of
DNA precursors.
Another biochemical property that has been useful in understanding the
in vivo function of a DNA polymerase is its fidelity. Because the
error specificity of a DNA polymerase reflects the way it interacts with its
substrates, investigation of error specificity may offer clues as to the DNA
transactions the enzyme is able to conduct. For example, the low fidelity and
the mutational specificity of DNA polymerases 
and 
, both members
of family Y, suggest that they participate in somatic hypermutation of
immunoglobulin genes (reviewed in Ref.
14), a process responsible for
generation of high affinity antibodies. Additional experimental results
support this hypothesis (14,
23,
24). Similarly, non-templated
additions are a signature of synthesis by TdT, implicating TdT in V(D)J
recombination, essential for diversification of immunoglobulin genes
(25). Also, analysis of the
fidelity of human Pol 
, responsible for the replication and repair of
the mitochondrial genome, showed that its mutational specificity is consistent
with the nature of mutations associated with aging and disease
(26). This finding supports
the hypothesis that accumulation of Pol errors leads to mitochondrial
dysfunction. Based on the logic that an error signature can be informative
regarding possible functions, we present here a detailed analysis of the
fidelity of Pol and discuss the implications for its possible
function in vivo.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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was described previously
(20).
Forward Mutation Assay—This assay scores errors in the
lacZ 
gene in M13mp2 during synthesis to fill a 407-nucleotide
gap (27). Reaction mixtures
(25 µl) contained 1 nM gel-purified M13mp2 gapped DNA substrate,
50 mM Tris-HCl (pH 8.5), 2.5 mM MgCl2, 1
mM dithiothreitol, 2 µg of bovine serum albumin, 4% glycerol,
and 10 µM each of dATP, dGTP, dCTP, and dTTP. Polymerization
reactions were initiated by adding Pol (300 nM) or Pol
(20 nM), incubated at 37 °C for 1 h, and terminated by
adding EDTA to 15 mM. Reaction products were analyzed by agarose
gel electrophoresis as described
(27). Correct synthesis
produces M13mp2 DNA that yields dark blue phage plaques upon introduction into
an Escherichia coli -complementation strain and plating on
indicator plates. Errors are scored as light blue or colorless mutant phage
plaques. DNA from independent mutant clones was sequenced to define the
lacZ mutation. Because most of the mutant clones generated by Pol
contained both phenotypically detectable and silent changes, the
error rates are described as the number of observed mutations divided by the
number of nucleotides sequenced. Error rates for Pol were calculated as
described (27).
Short Gap Frameshift Reversion Assay—Construction of the DNA
substrate in which the 6-nucleotide gap contains a portion of the
lacZ -complementation sequence modified by the introduction of
a TTTT sequence has been described
(28). Because of the
additional nucleotides, the resulting template encodes a colorless M13 plaque
phenotype. Frameshift mutations that restore the reading frame result in blue
plaques. Gap-filling reaction mixtures (20 µl) contained 50 mM
Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM
dithiothreitol, 2 µg of bovine serum albumin, 4% glycerol, 1.6
nM gapped DNA, 500 µM each of dATP, dGTP, dCTP, and
dTTP, 400 units of T4 DNA ligase, and 100 nM Pol or 50
nM Pol . After a 1-h incubation at 37 °C, reactions were
terminated by adding EDTA to 15 mM; the products were separated on
an agarose gel. The covalently closed circular DNA products were electroeluted
from gel slices, and the DNA was precipitated with ethanol. DNA products were
introduced into E. coli by electroporation and followed by plating as
described (27).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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in the Forward Mutation
Assay—An earlier study indicated that the fidelity of Pol
was similar to that of Pol for single-base substitution errors at a TGA
codon in the lacZ gene
(20). To see if a more
comprehensive view of the fidelity of Pol offered clues to its
function, we used the M12mp2 forward mutation assay that detects a broad range
of substitution, deletion, and addition errors in a large number of sequence
contexts (Table I). This assay
requires the enzyme to fill in a 407-nucleotide gap in an M13mp2
double-stranded circular DNA substrate. Pol was able to conduct
complete gap-filling, as determined by DNA product analysis in an agarose gel
(not shown, but see Fig. 3 in Ref.
27, for a typical result). The
gap-filled DNA products were introduced into E. coli cells and
plated, and the plates were scored for total and mutant M13mp2 plaques (see
"Experimental Procedures"). As a control, we carried out reactions
with human Pol . DNA synthesis by Pol generated lacZ
mutants at a frequency of 3.5%, consistent with previously reported values
(28). Using the same reaction
conditions, Pol generated mutants at a 6-fold higher frequency (21%,
as observed in several independent determinations), indicating that Pol
is less accurate than Pol .
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Error Specificity—We sequenced DNA isolated from 103
independent lacZ mutants to determine the error specificity of Pol
. Single-base substitution, deletion, and insertion errors were
observed (Table I) and they
were distributed throughout the template DNA present within the gap
(Fig. 1). In addition, a few
multiple-base changes were observed. The calculated error rates for the
different types of errors made by Pol are presented in
Table II, and
Fig. 2 shows average
single-base error rates in comparison to other exonuclease-deficient mammalian
DNA polymerases examined with this same assay and template. These data reveal
that Pol has the lowest single-base deletion fidelity of any of these
DNA polymerases, and that its average single-base deletion error rate exceeds
its single-base substitution error rate. Further, they show that Pol
is, on average, 32-fold less accurate for single-base deletions than is
homologous Pol .
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In contrast, the overall base substitution error rate of Pol is
closer to what was observed for Pol . Pol generated both
transitions and transversions (Fig.
1, Table III), with
the main error (13 of 38) being formation of a T·dGMP mispair (error
rate of 14 x 10–4). The preference for this
mispair has also been observed previously with other polymerases
(29,
30). Absent from the Pol
spectrum (Fig. 1) are
T to G transversions at template base 103. This error is the most prominent
feature of Pol base substitution error spectra
(28,
31) and has been suggested to
result from a dislocation mechanism
(13,
32). According to this
mechanism, a correct insertion may occur on a transiently misaligned
template-primer, followed by realignment to create a mispair that is extended
to create a base substitution. Although we have no indication of dislocation
mutagenesis by Pol at this particular site, the specificity of 22 of
the 38 Pol -generated base substitutions shown in
Fig. 1 is consistent with the
possibility that some of these could result from dislocation.
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The Frameshift Error Specificity of Pol —To
consider possible mechanisms for the high single-base deletion error rate of
Pol , we analyzed the sequence contexts in which these deletions
occurred. The majority of –1 base deletions occurred at iterated
nucleotides (Fig. 1). Deletion
of iterated pyrimidines was more frequent than deletions of iterated purines,
which could reflect weaker stacking interactions between adjacent template
pyrimidines than between adjacent template purines. Deletion of nucleotides in
repetitive sequences could result from classical template-primer slippage, a
signature of which is an increase in error rate with an increasing number of
repeat units in the repetitive sequence
(33). Therefore, we looked at
the relationship between the error rate and homopolymeric run lengths for the
single-base deletions generated by Pol . The results
(Fig. 3) indicate a 6-fold
increase in error rate for deletions in a two-nucleotide homopolymeric run as
compared with the rate of deletions of non-iterated bases. Interestingly, no
additional increase in error rate is observed as the run length increases
further. This is in contrast to the results with Pol , where deletion
rates are lower (in Fig. 3,
note difference in scale on y axis), and there is a more regular
increase in error rate with increasing run length.
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Low Frameshift Fidelity during Short Gap Filling Synthesis—A
number of observations with other DNA polymerases reveal a correlation between
single-base frameshift error rates and the processivity of DNA synthesis (see
Ref. 34). This has led to the
notion that a misaligned intermediate is more likely to form when a polymerase
dissociates and/or reassociates with the template-primer. We have reported
previously that Pol is distributive when copying an open
template-primer such as the 407-nucleotide gap used in the forward mutation
assay but is more processive when filling a 5-nucleotide gap in which the
5' end is phosphorylated
(20). Therefore, we tested
whether the single-base deletion fidelity of Pol might be higher when
processively filling a short gap. The assay
(28) detects single-base
deletions (as dark blue revertant plaques) within a 6-base gap containing the
template sequence 5'-CTTTTA (see "Experimental Procedures").
In this assay, the frequency of Pol -generated revertants is also
high, over 100-fold higher than that of Pol
(Table IV). Thus, even when
filling a short gap, Pol produces deletions at an unusually high
rate. DNA sequence analysis confirmed that 39 of 40 dark blue revertants
lacked one of the four Ts, whereas only one resulted from a deletion of the
template A. This result is consistent with the Pol specificity
revealed in the forward assay (Fig.
1).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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is unusual in that its average single-base frameshift error rate is
very high and exceeds its average rate of base substitutions. This finding has
interesting functional and mechanistic implications. Pol has been
implicated in base excision repair because, like Pol , it possesses a
dRP lyase activity in its 8-kDa domain, it shows a preference for filling
short-gap substrates with a 5'-phosphate, and it can substitute for Pol
in a single-nucleotide base excision repair reaction in vitro.
Despite these shared features and similar base substitution fidelity, the
average single-base deletion fidelity of Pol is over 30-fold lower
than that of Pol (Table
II). In fact, the average –1 base deletion error rate of Pol
is even higher than those of polymerases of the Y family, which are
renowned for their infidelity. Because of the increase in the single-base
deletion rate for runs as compared with non-iterated nucleotides, some of
these deletions may reflect classical strand slippage
(33). However, the relatively
high Pol deletion rate for non-iterated nucleotides
(Fig. 3) and the lack of a
further increase in rate with increasing run length suggests the involvement
of additional mechanisms and/or unusual interactions of Pol with its
substrates. Possibilities include misalignment in the polymerase active site
(35–39),
nucleotide misinsertion followed by primer relocation
(32,
40), or some other mechanism.
The fact that the relationship between single-base deletion rates and
homopolymeric run length are different for Pol compared with Pol
(Fig. 2) or with other
polymerases, including the Y family members Pol and Pol 
(41,
42), suggests that Pol
interacts with the DNA substrate differently than those other
enzymes. The high deletion error rates and lack of an increase in rate for
runs of three or more as compared with a run of two imply that Pol is
relatively efficient at utilizing misaligned DNA substrates stabilized even by
only one correct base pair at the primer terminus.
The higher frameshift rate of Pol as compared with Pol
indicates differences in the interactions that control the proper alignment of
the template-primer and the incoming dNTP. These differences were not
anticipated because, as shown in Fig.
4, many of the Pol residues that comprise the nascent base
pair binding pocket are conserved in Pol . Among these is Arg-283
(Arg-517 in Pol ), which contributes to the fidelity of Pol by
providing interactions that stabilize the templating base. Mutations at this
residue result in Pol with greatly reduced base substitution
(43) and frameshift fidelity
(44). In fact, the single-base
frameshift error rates measured for the Arg-283 Ala derivative of Pol
are similar to those observed for wild-type Pol . Thus,
although the presence of an arginine residue at this position in Pol
(Arg-517) suggests that the function of this side chain is likely to be
conserved, it is nonetheless possible that slight differences in the
orientation of this residue could affect the interactions that control the
alignment of the templating base with the incoming dNTP, contributing to the
low frameshift fidelity of the enzyme. In addition, the ability to form and/or
to utilize misaligned template-primers could be affected by or linked to any
of the three residues in the nascent base pair binding pocket of Pol
that are not conserved in Pol : Ala-185, Lys-280, and Asp-276
(Fig. 4). Ala-185 (Lys-422 in
Pol ) is located in proximity to the phosphates of the incoming
nucleotide, and Lys-280 (Arg-514 in Pol ) and Asp-276 (Ala-510 in Pol
) stack with the templating base and the base of the incoming dNTP,
respectively (Fig. 4).
Interestingly, the presence of an alanine residue at the position
corresponding to Asp-276 in Pol is believed to contribute to the high
affinity of Pol for dNTPs
(20).
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The frameshift fidelity of Pol does not increase when processively
filling a short gap (Table IV),
suggesting that misaligned frameshift intermediates may be efficiently formed
and/or stabilized within the active site of the enzyme and extended even when
it is conducting processive synthesis. In this respect, Pol resembles
Pol . It has been suggested that during short gap synthesis, the
polymerase domain of Pol may dissociate from and then reassociate with
the template-primer, allowing its misalignment, while the enzyme remains
tethered to the DNA by the binding of the 8-kDa domain to the 5' end of
the gap (28). Pol ,
the processive synthesis of which in a short gap is also facilitated by the
presence of a 5'-phosphate on the downstream duplex, may behave in a
similar way. The recent solution structure of the 8-kDa domain of Pol
(45) has confirmed the
conservation of the residues involved in 5'-phosphate binding within a
positively charged DNA binding groove. This positively charged surface in the
8-kDa domain of Pol is significantly larger than in Pol . It
has been suggested that this larger electrostatic surface may allow Pol
to more stably bind the 5'-phosphate, thus limiting strand
displacement synthesis
(20).
Is the propensity of Pol to use misaligned template-primers
related to its physiological function? One possibility is that Pol
may be involved in NHEJ, which along with homologous recombination, is one of
two pathways responsible for the repair of double-strand breaks (DSBs) in
eukaryotic cells (46,
47). During NHEJ, broken DNA
ends can be aligned using minimal base pairing (microhomology), creating
imperfect duplexes with short gaps that need to be filled by a DNA polymerase.
By virtue of its ability to use substrates with minimal homology at 3'
ends (Fig. 3), human Pol
may perform this reaction. Two other members of the Pol X family have
already been implicated in DNA end-joining reactions, Pol µ and TdT. TdT is
involved in and restricted to repair of DSBs during V(D)J recombination,
whereas Pol µ, which has a much wider tissue distribution
(9), has been suggested to
function in a general NHEJ pathway
(17,
18,
48). Pol µ associates with
Ku, a key component of the end-joining reaction, and forms a stable complex on
DNA in the presence of Ku and ligase IV/XRCC4, another core NHEJ factor. Such
a complex is essential for an efficient end-joining reaction involving
alignment of ends, gap filling, and ligation
(17,
49). Consistent with this
role, Pol µ can promote the formation and extension of misaligned
primer-templates (Ref.
18).2 The present
study suggests that Pol , with its unusually high rate for
misalignment-mediated errors and its preference for short gap substrates, also
appears to be well suited to participate in NHEJ. The possible involvement of
Pol in this process is further suggested by genetic
(50) and biochemical studies
(51) indicating that in yeast,
end-joining depends upon the function of DNA Pol IV, a close homologue of
human Pol . In a recent study, Heidenreich et al.
(52) showed that NHEJ
contributes to mutagenesis in non-replicating diploid yeast cells. They
suggested that this mutagenic process might not involve a DNA polymerase.
Nevertheless, we find it intriguing that the majority of mutations observed in
that study were single-base deletions in short homopolymeric runs. This
specificity is characteristic of Pol and thus suggests the
involvement of Pol IV. The end-joining activity of Pol IV requires the
presence of its N-terminal BRCT domain
(50,
51). It has been shown that
through the BRCT domain, Pol IV interacts with Dnl4, a subunit of the
Dnl4-Lif1 complex (51), which
is the homologue of the human ligase IV-XRCC4 complex. Thus, the BRCT domain
may be involved both in the recruitment of the polymerase and in interactions
that couple the gap-filling and the ligation steps. Pol , like Pol IV,
Pol µ, and TdT also has an N-terminal BRCT domain, which appears to be a
common feature of polymerases involved in NHEJ. Finally, a recent study has
revealed that Pol has TdT activity and the capacity to elongate RNA
primers (53), both of which
could perhaps be related to its functions in vivo.
In yeast, the majority of DSBs are repaired by homologous recombination.
However, in higher eukaryotes, repair of DSBs is mostly dependent on the NHEJ
pathway (47). Homologous
recombination is restricted to late S and G2 phases of the cell
cycle, whereas NHEJ is the predominant repair mechanism during G0,
G1, and early S phases
(54). It is worth noting that
while the cellular dNTP pools are highest during S and G2 phases,
they are lowest in G0
(55), which is consistent with
the hypothesis that DNA Pol may be involved in DNA transactions
occurring when the concentration of precursors is low
(20). Hence, Pol is a
good candidate to function throughout the cell cycle in DNA transactions that
involve NHEJ. Its intrinsic dRP lyase activity
(22) implies that Pol
may be useful for repair of DSBs with certain types of damaged DNA ends,
e.g. those resulting from abortive processing by base excision repair
enzymes of clustered DNA lesions composed of abasic sites, oxidized bases, and
strand breaks caused by ionizing radiation
(56).
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FOOTNOTES |
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These authors contributed equally to this work.
|| To whom correspondence should be addressed. Tel.: 919-541-2644; Fax: 919-541-7613; E-mail: kunkel{at}niehs.nih.gov.
1 The abbreviations used are: Pol, polymerase; TdT, terminal
deoxynucleotidyltransferase; dRP, 5', 2'-deoxyribose 5-phosphate;
NHEJ, nonhomologous end-joining; DSB, double-strand break; BRCT, breast cancer
susceptibility gene 1 C terminus.
2 J. F. Ruiz, K. Bebenek, M. Garcia-Diaz, L. Blanco, and T. A. Kunkel,
unpublished data.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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" immediately above the added
base. The arrow indicates the direction of synthesis.
