DNA Repair 4 (2005) 1358–1367

Structure–function studies of DNA polymerase lambda

Miguel Garcia-Diaz, Katarzyna Bebenek, Guanghua Gao, Lars C. Pedersen,
Robert E. London, Thomas A. Kunkel ∗
Laboratory of Structural Biology and Laboratory of Molecular Genetics NIEHS, NIH, DHHS, Research Triangle Park,
NC 27709, USA
Available online 4 October 2005

Abstract
DNA polymerase lambda is a member of the X family of polymerases that is implicated in non-homologous end-joining of double-strand breaks
in DNA and in base excision repair of DNA damage. To better understand the roles of DNA polymerase lambda in these repair pathways, here
we review its structure and biochemical properties, with emphasis on its gap-filling polymerization activity, its dRP lyase activity and its unusual
DNA synthetic (in)fidelity.
Published by Elsevier B.V.

Keywords: DNA polymerase lambda; Family X; Non-homologous DNA end-joining; DNA repair; dRP lyase; Enzyme mechanism

1. Introduction template–primer. The low processivity of Pol ␤ suggested a role

To maintain genome stability over many generations, DNA
based organisms must not only replicate their genomes accu-
rately, they must also remove damaged nucleotides and replace
them with undamaged nucleotides via DNA synthesis [1]. Repli-
cation and repair DNA synthesis in cells is conducted by DNA
polymerases that are remarkable both in number, e.g., 17 poly-
merases are encoded by the human genome [2], and in functional
diversity. Over two decades ago Braithwaite and Ito [3,4] classi-
fied all DNA polymerases known at that time into four families
according to their primary sequence. Families A, B, and C
consisted of enzymes homologous to the Escherichia coli poly-
merases I (PolA), II (PolB) and III (PolC), respectively.
However, neither eukaryotic DNA polymerase beta (Pol ␤)
nor terminal deoxynucleotidyl transferase (TdT) showed sub-
stantial homology to the E. coli enzymes. For this reason, and
because their sequences were surprisingly similar to each other,
these polymerases were included in a newly designated X fam-
ily.
Both family X DNA polymerases were unorthodox. TdT
disregarded the use of a template strand to instruct incorpo-
ration, and Pol ␤ had very low processivity, i.e., the ability to
incorporate multiple nucleotides without dissociating from the
∗ Corresponding author. Tel.: +1 919 541 2644; fax: +1 919 541 7613.
E-mail address: kunkel@niehs.nih.gov (T.A. Kunkel).
in short patch DNA repair synthesis. It was later shown that Pol
␤ is the major mammalian enzyme participating in base exci-
sion repair (BER) [5–7]. In contrast, TdT randomly incorporates
nucleotides at DNA ends during V(D)J recombination, a process
that generates variability in antigen receptor genes during the
differentiation of lymphocytes [8]. Although TdT and Pol ␤ are
vertebrate enzymes, an enzyme with properties similar to those
of Pol ␤ was later identified in Saccharomyces cerevisiae and
designated Pol IV [9,10]. When the gene encoding Pol IV was
cloned and sequenced, it was 26% identical and 50% similar
to rat and human Pol ␤ [10]. Nonetheless, Pol IV was substan-
tially larger than Pol ␤ and contained an additional N-terminal
domain, later identified as a BRCT domain [11,12]. Although
dispensable for BER [13,14], yeast Pol IV was shown to partic-
ipate in the gap-filling step of the non-homologous end-joining
(NHEJ) pathway of double-strand break repair [15].
These observations raise the question of how related family
X polymerases have quite different physiological roles. A
partial explanation came with the discovery of two new
members of family X, DNA polymerases ␭ [16–18] and ␮ [19].
Both are homologs of Pol ␤, but like Pol IV, they also contain
BRCT domains. Pol ␮ and Pol ␭ have been identified in several
organisms, including plants [16,20]. Other Pol ␤ homologs
have also been identified in bacteria, archaea, protozoa and
viruses (see Table 1 and Fig. 1). Although not the subject of
this review, numerous other DNA polymerases have also been
identified since the original classifications of Braithwaite and

1568-7864/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.dnarep.2005.09.001
 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367 1359

Table 1
Family X DNA polymerases
Polymerase Model organism Biochemical properties dRP lyase BRCT domain Biological functions

Mammalian Pol ␤ H. sapiens DNA-dependent activity, gap-filling, AP lyase, Yes No BER

PCNA interaction
Mammalian Pol ␭ H. sapiens DNA-dependent activity, gap-filling, high dNTP Yes Yes NHEJ, BER?
affinity, low indel fidelity, PCNA interaction, can
utilize ribonucleotide primers
Mammalian Pol ␮ H. sapiens DNA-dependent activity, low fidelity, No Yes NHEJ, V(D)J recombination
template-independent activity, ribonucleotide
insertion
TdT (vertebrates) M. musculus Template-independent activity, ribonucleotide No Yes V(D)J recombination
insertion
Pol IV S. cerevisiae DNA-dependent activity, gap-filling, low fidelity, Yes Yes NHEJ, BER?
ribonucleotide insertion
S. pombe ? ? Yes NHEJ?
Plant Pol ␭ A. thaliana ? ? Yes DNA repair?
O. sativa DNA-dependent activity, PCNA interaction Yes Yes DNA repair?
Protozoan Pol ␤ C. fasciculata DNA-dependent activity Yes No Mitochondrial DNA repair?
T. brucei DNA-dependent activity Yes No Mitochondrial DNA repair?
L. infantum ? ? Nuclear DNA repair?
Other Pol X Some archea and ? ? No ?
bacteria
Viral Pol X ASFV DNA-dependent activity, gap-filling, AP lyase No No DNA repair?
MSEPV ? ? No BER?

Ito [3,4]. These include members of the widespread family Y
[21], comprising enzymes with the ability to perform lesion
bypass [2,22,23], and several members of family D, composed
of archaeal enzymes [24].
2. Structural organization of Pol ␭
Like other family X polymerases, Pol ␭ is a relatively
small, single-subunit enzyme lacking a 3 → 5 exonucleolytic
activity [16–18,25]. The 575 amino acid polypeptide of human
Pol ␭ comprises an N-terminal BRCT domain, believed to
mediate protein–protein interactions, the catalytic core and a
serine-proline rich region connecting the two (Fig. 2A). The
serine-proline rich region is present in Pol ␭ and its yeast
homologue Pol IV, and has been suggested to be a target for
posttranslational modifications [16]. The sequence of the 39 kDa
catalytic core of Pol ␭ is most similar to human Pol ␤ (33%
amino acid identity) and the catalytic core of Pol ␮ (31% amino
acid identity) and shares the modular organization characteristic
of X family enzymes (Fig. 2A). The catalytic core is composed
of an N-terminal 8 kDa domain (purple) unique to family X poly-
merases and a polymerase domain, including the (blue) fingers,
(red) palm containing the catalytic carboxylates, and the (green)
thumb subdomains that are common to all polymerases [26].
The 2.1 Å resolution X-ray crystal structure of the 39 kDa cat-
alytic domain of human Pol ␭ in complex with a template–primer
containing a two-nucleotide gap (Fig. 2B) reveals a general fold
like that of Pol ␤ and TdT and indicates that Pol ␭ shares the
same secondary structure elements [27]. The catalytic core of
Pol ␭ has two DNA binding sites that impose a 90◦ bend in the
DNA, exposing the 3 primer terminus. The polymerase domain
binds the primer terminal base pair and the upstream duplex,
while the 8 kDa domain binds the DNA downstream of the gap.
The interactions between the polymerase domain and the duplex
DNA upstream of the primer terminus are not extensive and do
not involve interactions with the bases beyond the primer termi-
nal base pair [27]. Furthermore, as indicated by the electrostatic
surface potential, the template-binding groove in Pol ␭ is not as
positively charged as in Pol ␤, suggesting weaker interactions
with the template strand. In contrast, the binding of the DNA
downstream of the gap is predominantly through the interaction
of the 5 terminus of the gap with the positively charged residues
of the 8 kDa domain. This binding is greatly strengthened by the
presence of a 5 terminal phosphate, which is held in a posi-
tively charged pocket that comprises the dRP lyase active site
(discussed below). The interactions with the 5 phosphate stim-
ulate the polymerase activity of Pol ␭ on gapped substrates [25].
3. Biochemical properties and activities
Pol ␭ is a template-dependent polymerase [18,25,28]. Under
certain conditions, Pol ␭ can perform template independent
incorporation [29], albeit with very low efficiency [30]. A simi-
lar behavior has been observed with Pol ␤ [31,32]. A preferred
substrate for Pol ␭ is a short gap with a phosphate on the 5 end, a
preference shared by at least some other family X polymerases.
Whereas Pol ␭ is distributive on a primed single-strand template,
its activity is stimulated during synthesis in a short gap contain-
ing a phosphate at the 5 end. Also, Pol ␭’s strand displacement
activity is limited by the presence of a phosphate at the 5 end
of the gap [25]. A putative PCNA binding motif has been recog-
nized as part of the HhH-motif in the Pol ␭ fingers subdomain,
consistent with the observations that Pol ␭ physically interacts
with PCNA and that PCNA influences its polymerase activ-
ity [28,33,34]. These observations suggest that PCNA might
1360 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367

Fig. 1. Evolutionary relationships between family X members. An unrooted phylogenetic tree built using a primary sequence alignment of a segment of the catalytic
domain. Different enzymes are grouped (shaded areas) into the five main enzyme classes in the family: Pol ␤, Pol ␭, Pol ␮, TdT, and trypanosomatid (Try) Pol ␤-like
enzymes.

modulate synthesis by Pol ␭ in vivo. While the function of the
proline-serine domain in Pol ␭ is not clear, it is reported to sup-
press the polymerase activity of the enzyme in vitro [28]. Pol ␭
has a high affinity for dNTPs [25], a property that can be partly
attributed to the presence of an uncharged side chain (Ala) at
residue 510, positioned against the base of the incoming dNTP
in the nascent base pair binding pocket [25]. The high affinity of
Pol ␭ for dNTPs may allow it to conduct synthesis when the con-
centration of precursors in the cell is low, e.g., outside S-phase in
cycling cells or in quiescent cells. Also, Pol ␭ has unusually low
fidelity and a unique error specificity. It is the only human DNA
polymerase studied to date whose average single base deletion
error rate exceeds its average single base substitution error rate.
Pol ␭ deletes single nucleotides at a rate that is much higher
than for Pol ␤. In fact, the Pol ␭ deletion rate exceeds those of
the notoriously inaccurate polymerases in the Y family [35]. In
contrast, Pol ␭ generates single base substitution errors at rates
that are only slightly higher than for Pol ␤ [25,35]. Both the
high rate and the specificity of Pol ␭-generated −1 deletions
reflect its ability to use template–primers with limited base pair
homology at the primer terminus. As discussed below, this has
both structural and functional implications. Interestingly, a high
rate for misalignment-mediated errors is also a characteristic of
yeast Pol IV [36], suggesting similarities between Pol ␭ and Pol
IV with respect to enzyme–substrate interactions that control
fidelity.
4. dRP lyase activity of the 8 kDa domain of Pol ␭
Single-nucleotide BER in mammalian cells begins with
removal of a damaged base by a DNA glycosylase, followed
by incision of the sugar–phosphate backbone 5 of the AP site to
 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367
Fig. 2. Domain and subdomain organization of Pol ␭. (A) Linear diagram indi-
cating the different domains that compose the full-length Pol ␭ protein: BRCT,
Ser-Pro rich domain (S-P), 8 kDa domain and the polymerase catalytic domain,
composed of Fingers, Palm, and Thumb subdomains. (B) Ribbon representation
of the 39 kDa catalytic core of Pol ␭ in complex with a two-nucleotide gap (the
DNA is shown in stick representation). The molecular surface is shown in trans-
parent gray. The different subdomains are color coded as in (A). The atomic
coordinates correspond to PDB 1RZT.
create a 5 terminal dRP group. The missing nucleotide is then
replaced by a DNA polymerase, the dRP group is removed and
the nick is ligated to complete BER [7,37]. It has been well
documented that the polymerase and dRP lyase activities of
mammalian Pol ␤ participate in single nucleotide BER [6]. Pol
␭ also has a dRP lyase activity that can remove a 5 terminal
sugar–phosphate (dRP) group [38]. Since Pol ␭ can fill in short
gaps, has a dRP lyase activity and can substitute for Pol ␤ in a
reconstituted in vitro BER reaction [38], it has been proposed
that it too may participate in some form of BER in cells. Support
for this hypothesis comes from two recent studies implicating
Pol ␭ in BER. Pol ␭ seems to be able to carry backup repair
in Pol ␤ −/− cells [39], an observation that correlates with the
observed sensitivity of Pol ␭ −/− fibroblasts to oxidative dam-
age [40].
The dRP lyase activity of Pol ␤ appears to be rate limiting
for BER [41–45]. The 8 kDa domain of Pol ␤ has been shown to
possess significant lyase activity—even when assayed as a sepa-
rate domain [46], and a similar activity has been observed for the
homologous domain of Pol ␭ [38]. This lyase domain is thus one
of the smallest proteins to exhibit significant enzymatic activ-
ity. However, it is worth noting that even short peptides such as
Lys-Trp-Lys are catalytic for the lyase reaction [47]. Elimination
of the abasic dRP group does not require a divalent metal ion.
This reaction has been suggested to proceed either by a hydrol-
ysis reaction, as occurs for most nucleases, or via an elimination
reaction that proceeds via formation of a Schiff’s base interme-
1361
diate [46]. Identification of an unsaturated enzymatic product
[46] and reductive trapping of Schiff’s base adduct intermedi-
ates [38,48,49] support a ␤-elimination pathway for Pol ␤ and
Pol ␭. The ␤-elimination mechanism probably represents a more
attractive alternative since it inherently selects for the reducing
sugar at the abasic site, while the hydrolysis mechanism lacks
such selectivity, and thus would need to be very carefully con-
trolled to prevent unwanted nuclease activity at other sites on
the DNA.
An evaluation of the proposed contributions of various
residues to the lyase reaction mechanism can be made based on
a structural alignment of the Pol ␭ and Pol ␤ domains [27,50].
Schiff’s base formation is viewed as proceeding from an open
chain aldehyde form of the sugar which is thought to represent
only ∼1% of the abasic deoxyribose, the remainder being in
the furanose form [51]. Given the low probability of the open
chain aldehyde, it has been suggested that opening of the ring
could result from protonation of the ring oxygen O-4 by residues
K68 [52,53] or K35 [53]. However, in the Pol ␭/Pol ␤ structural
alignment [27,50], both of these residues are replaced by argi-
nine, which has a much higher pK and is thus unlikely to serve
as a proton donor. Similarly, a proposed role for His34 of Pol
␤ acting as a general base in the removal of a hydroxyl pro-
ton [52] also is inconsistent with the presence of tryptophan
at the corresponding position in Pol ␭ [50]. A possible con-
tribution of E26 in the abstraction of the ribose C-2 proton
from the Schiff’s base intermediate [53] also would need to be
modified since E26 is structurally aligned with Y267 in Pol ␭.
These residue substitutions do not necessarily rule out the earlier
proposals, but it becomes necessary either to postulate that the
two structurally homologous domains function with significant
mechanistic differences, or to modify the mechanism and/or par-
ticipating residues. Uncertainty in the roles of various residues
has resulted in part from the lack of a detailed structure showing
the catalytic position of the abasic sugar or a stable analog.
The open chain form of the deoxyribose phosphate is then
available for reaction with an active site nucleophile, identified
as K72 in Pol ␤ [48,49,52–54] or the structurally homologous
K312 in Pol ␭ [38]. Although the central role of Pol ␤ K72 in
the lyase reaction has been confirmed by mutagenesis studies
[48,54] and its involvement in Schiff’s base chemistry by reduc-
tive trapping with borohydride [48,49], the larger question of
how a lysine residue with a high pK value can be optimized in
order to function as a nucleophile for Schiff’s base formation
remains unanswered. Studies with the model tripeptide KWK
demonstrate that it is the lower pK ␣-amino group of K1, rather
than the higher pK ␧-amino group of the lysines that is primarily
involved in the lyase reaction [47]. In other cases, for example, E.
coli Fpg protein, the lyase reaction involves the N-terminal pro-
line residue [55] so that again, a low pK amino group is utilized
for this type of chemistry. Consistent with expectations, we have
recently labeled the lyase domain of Pol ␭ with [␧-13 C]lysine
and obtained a pK value of 9.8 for K312 based on titration of the
resonance shift [Gao et al., in preparation]. This was the lowest
pK value measured for any of the 10 lysine residues in the Pol ␭
lyase domain, but is still nearly three orders of magnitude above
neutrality.
1362 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367

Fig. 3. Possible roles of K312 and Y279 in the dRP lyase reaction. Enzyme residues are indicated in blue, and the substrate in black. Initial proton transfer to form
the aldehyde creates the unprotonated lysine nucleotide which is stabilized by a hydrogen bonding interaction with Y279. It then reacts with C-l of the abasic
deoxyribose to form the Schiff’s base. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

We are, therefore, faced with the fundamental dilemma of a
rate-limiting step in the DNA repair process involving a lyase
domain demonstrated to be principally responsible for this reac-
tion that does not appear to be well optimized for the proposed
reaction chemistry. There are several possible resolutions to this
mechanistic dilemma. (1) The lysine pK value in the active
complex may be significantly lower than is observed for the
uncomplexed domain. However, since both the terminal and
the penultimate phosphate groups would be expected to form
salt bridges with the basic residues in the active site, it is diffi-
cult to envision how DNA binding would lower the lysine pK
value. Indeed, in the 1RZT structure [27] there is an apparent
salt bridge between K312 and the terminal phosphate. (2) An
attractive mechanistic solution would combine the ring opening
function with Schiff’s base formation, so that protonation of O-
4 by K312 simultaneously creates the necessary (unprotonated)
nucleophile (Fig. 3). Some support for this idea is suggested by
the close proximity of the ␧-amino group of K312 to the hydroxyl
group of Y279. The tyrosyl hydroxyl group could then act as an
H-bond acceptor from the protonated lysine, and a hydrogen
bond donor to the unprotonated lysine, helping to stabilize the
nucleophilic NH2 form until it reacts with C-1 . In fact, it is inter-
esting to note that this tyrosine residue is conserved in all family
X enzymes shown to possess a dRP lyase activity (including Pol
␤, Pol ␭, Pol IV and the Pol ␤ from C. fasciculata [56]), while it is
substituted by a phenylalanine in the family X members known
to lack this activity (i.e., Pol ␮ and TdT). (3) A third possibility
would involve facilitation of the reaction by another molecule,
which could help by affecting the structure of the Pol ␭ complex
with the abasic site, altering the environment of K312 and hence
its pK, or even by supplying another low pK amino group for
the reaction. In this context, we note that the AP endonuclease
Apel has recently been shown to significantly accelerate the dRP
lyase reaction of Pol ␤ [57]. Additional structural studies of the
lyase domain will be required to resolve these questions about
its activity.
5. Mechanism of polymerization
Structural characterizations of Pol ␤ and DNA polymerases
in families A and B suggest that dNTP binding induces large
conformational changes in the relative positions of the fingers
and thumb subdomains [58–60]. For some polymerases, this
change from an open to closed conformation may correspond
to the kinetically determined rate limiting step for incorporation
and thus contribute to the nucleotide selectivity of the reaction
[26,61,62]. This and other conformational changes might serve
as multiple fidelity checkpoints [63] as the active site and the
nascent base pair binding pocket are assembled. The nascent
base pair is accommodated between the primer terminal base
pair and a small number of amino acid residues that form the
other walls of the binding pocket. For polymerases in fami-
lies A, B, and X, steric complementarity in this binding pocket
has been suggested to be a general mechanism by which DNA
polymerases select the four correct and geometrically equiva-
lent correct Watson–Crick base pairs and discriminate against
non-Watson–Crick base pairs with different shapes [61].
Unlike Pol ␤, structural characterization of Pol ␭ indicates
that the catalytic cycle does not involve major subdomain
motions, i.e., Pol ␭ is in a closed conformation prior to dNTP
binding [64]. Instead, dNTP binding induces a major shift in
the positioning of the template strand of the DNA. This DNA
conformational change is accompanied by protein conforma-
tional changes in a ␤-hairpin in the palm subdomain, a loop in
the thumb subdomain, and a few side chains at and near the
active site. These changes establish protein interactions with
 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367
Fig. 4. Minor groove interactions near the active site of Pol ␭. Three Pol ␭
residues that are part of the nascent base pair binding pocket, Asn513, Arg517,
and Tyr505 (yellow) are involved in establishing interactions with the minor
groove of the DNA duplex (see text). The template strand is shown in dark
brown, the primer strand is olive, and the incoming triphosphate is magenta.
Postulated hydrogen bonds are shown as green dotted lines. The coordinates
correspond to PDB 1XSN. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of the article.)
the DNA minor groove that are likely to be crucial for cataly-
sis and nucleotide selectivity. Unlike for the major groove, the
stereochemical nature of the minor groove is similar for each
of the Watson–Crick base pairs, and thus these minor groove
interactions are used by DNA polymerases to check for correct
Watson–Crick geometry. Such interactions have been observed
in family A [60,65,66] and B [67] enzymes, but appear to be
absent in DNA polymerases from family Y. In the case of Pol
␭, the dNTP induced conformational changes establish minor
groove interactions only with the terminal base pair and with the
templating base (Fig. 4). In addition, a protein residue (Asn513)
interacts with the minor groove of incoming nucleotide (Fig. 4).
These interactions are fewer than for family A and B enzymes
that contact several base pairs upstream of the primer terminus.
This fact, and the lack of extensive interactions with the template
strand, suggests that Pol ␭ has strict geometric requirements only
for the terminal base pair and the nascent base pair, the only two
base pairs directly involved in catalysis. This minimal require-
ment makes Pol ␭ well suited for participation in NHEJ (see
below).
The polymerization reaction requires a nucleoside triphos-
phate, a DNA substrate and divalent metal ions [68]. The study
of the stereochemistry of the polymerization reaction began with
the use of chiral nucleotide analogs [69]. Those studies sug-
gested that polymerization involves inversion of configuration
of the ␣-phosphate of the incoming nucleotide, and thus that
the reaction was likely to involve an inline attack of the 3 -O
on the ␣-phosphate [70]. This hypothesis has been supported
by crystal structures of DNA polymerases in a pre-catalytic
state, where the primer terminus lacks a 3 -OH group and is,
therefore, unable to react, thus trapping the incoming nucleotide
immediately before catalysis [71]. Those structures illustrating
1363
the geometry at the active site before the reaction takes place
have been key to understanding the role of the catalytic metal
ions and the essential active site residues [26]. Fig. 5 shows
a scheme of the Pol ␭ polymerization reaction, as suggested
by such a pre-catalytic complex. As is the case for other DNA
polymerases, three carboxylic residues (Asp427, Asp429, and
Asp490) are suggested to coordinate with two divalent metal
ions, thereby positioning the substrates and stabilizing the tran-
sition state [26]. A proposed catalytic metal (or metal A) helps
stabilizing the deprotonated 3 -O, while a dNTP binding metal
(or metal B) presents an ␣,␤,␥-tridentate coordination with the
phosphates of the incoming dNTP (Fig. 5B).
For most polymerases, the fate of the reactive groups after
catalysis has so far eluded structural characterization. With Pol
␭, however, it was possible to capture a post-catalytic complex
where the protein conformation was identical to that in the pre-
catalytic complex, with the only difference between the two
being related to the making and breaking of the phospohodiester
bond. The post-catalytic complex remains in a closed conforma-
tion and even contains inorganic pyrophosphate, the product of
the reaction. This allows comparison of the pre-catalytic and
post-catalytic complexes to envision the stereochemistry of the
reaction, with in-line attack on the ␣-phosphate by the 3 -O
resulting in phosphoryl transfer, the generation of pyrophos-
phate and an inversion of configuration of the phosphorus of the
␣-phosphate (Fig. 6).
Since the active site of Pol ␭ is constitutively active because,
unlike for other polymerases, its assembly is not dependent upon
a global conformational change, catalysis will only depend on
the correct positioning of the substrates, namely the 3 -O and the
␣-phosphate. This, therefore, should be favored by the enzyme
for correct incorporations but, in order to maintain fidelity of
synthesis, should be prevented for incorrect incorporations. The
mechanism by which the enzyme discriminates between correct
and incorrect insertions is likely related to the subtle active site
conformational changes. Comparison of Pol ␭ complexes before
and after dNTP binding suggests that the establishment of minor
groove interactions by the enzyme plays a significant role in
facilitating catalysis. The DNA shift, presumably necessary for
dNTP binding, together with the minor groove interactions of the
Tyr505 and Arg517 side chains (Fig. 4), results in a significantly
different conformation of the last base of the primer, where the
3 -O (absent in the pre-catalytic structure) would be closer to
its catalytic position. These minor groove interactions seem to
be sufficient to properly orient both bases in the terminal base
pair in the absence of a 3 -O, but the appropriate ring pucker
of the sugar of the primer terminus might depend on interac-
tions of this group with an active site metal or residue (Fig. 6).
Minor groove interactions could also contribute to nucleotide
selectivity. Because the position of the reactive groups seems
to be exquisitely modulated by the enzyme, the reaction could
presumably be very sensitive to any alteration in the geometry
of either the terminal base pair or the nascent base pair. Thus,
base damage or the presence of non-Watson–Crick base pairs
might result in a non-catalytic positioning of the 3 -O or the ␣-
phosphate. This is easily understood for the terminal base pair,
since minor groove interactions are established with both bases
1364 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367

Fig. 5. Postulated polymerization mechanism. (A) Scheme of the proposed reaction mechanism. Two divalent metal ions coordinate the reactive groups. A dNTP
binding metal (or metal B) establishes an ␣,␤,␥-tridentate coordination with the phosphates of the incoming nucleotide, while the second (catalytic or A) metal
coordinates the ␣-phosphate of the incoming nucleotide and the 3 -O. Both metals are postulated to stabilize the transition state, facilitating the nucleophilic attack
of the 3 -O on the phosphorus of the ␣-phosphate. (B) Coordination of the dNTP binding metal (metal B). The coordination of the Mg2+ ion (green) is shown in
green lines. It involves the three phosphates of the incoming dNTP, two aspartate residues (A427 and Asp429) and a water molecule (red). A simulated annealing
Fo–Fc omit electron density map, contoured at 2␴, is shown (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the
web version of the article.)

in that base pair, thus presumably discriminating against mis-
matched termini. A similar mechanism might be discriminating
against incorrect incorporations since, as minor groove interac-
tions determine the conformation of the templating base, binding
of a mismatched dNTP might result in an incorrect position of
the ␣-phosphate.
6. Pol ␭ and non-homologous end joining
In addition to its possible involvement in BER, Pol ␭ has been
implicated in NHEJ [72–74], a major pathway in multicellular
eukaryotes for repair of DSBs [75,76]. During NHEJ the bro-
ken, largely incompatible DNA ends may be aligned with the
use of limited base pairing. This may create imperfect duplexes
with small gaps that need to be filled by a DNA polymerase
prior to ligation [77]. Three other family X polymerases, TdT
[78], Pol ␮ [79] and yeast Pol IV [15,80] have been implicated
in this process, and Pol ␭ appears to be yet another family X
polymerase suited to perform this function. It has been demon-
strated that Pol ␭, like TdT and Pol ␮, physically interacts with
the key end joining factors Ku and XRCC4-Lig IV [73,74,81].
Similar to what was observed with Pol IV, Pol ␭’s polymerase
activity is stimulated by the interaction with the ligase complex
[73]. Pol ␭ can perform gap filling in a reconstituted in vitro
end joining reaction and in extracts of HeLa cells, and both its
physical and functional interactions with the end-joining fac-
tors are dependent on the presence of its BRCT domain [72,81].
Whereas, the role of TdT in repair of DSBs is restricted to V(D)J
recombination, the role of Pol ␮ and Pol ␭, which have a much
wider tissue distribution, is not yet completely defined. Studies
in cultured cells have demonstrated that Pol ␮ is involved in
V(D)J recombination, promoting accuracy during Ig light chain
recombination, while Pol ␭ is not [81]. This observation is con-
sistent with the finding that Pol ␮ but not Pol ␭ knockout mice
have a B-cell deficiency associated with more extensive dele-
tions during V(D)J recombination of light chain loci [82]. In
contrast NHEJ in extracts of HeLa cells appears to require only
Pol ␭ and not Pol ␮ [72]. The fact that Pol ␭ has a dRP lyase
activity suggests that perhaps it is involved in repair of DSBs of
damaged DNA ends. Thus, Pol ␮ may function in V(D)J recom-
bination, whereas, the function of Pol ␭ could be in general
NHEJ.
 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367
Fig. 6. Polymerization involves an inversion of configuration at phosphorus.
Overlay of a pre-catalytic (gray) and a post-catalytic (green) complex of Pol
␭. The nucleophilic attack of the 3 -O on the ␣-phosphate (P␣) results in bond
formation and an inversion of configuration at the phosphorus atom. The three
catalytic aspartates and the observed metal ion (dNTP binding metal) are shown.
The line of transfer is shown in orange. Hydrogen bonds are shown as dotted
lines. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
The ability of Pol ␭ to participate in NHEJ is clearly depen-
dent on the presence of its BRCT domain, which enables the
enzyme to interact with other end-joining proteins. However,
besides this basic requirement, there is growing evidence sug-
gesting that NHEJ polymerases have special properties enabling
them to conduct synthesis on the non-canonical substrates that
are encountered during NHEJ repair. For instance, an important
feature of gap filling in the context of NHEJ may be the capacity
of the Pol ␭ 8 kDa domain to bridge the gap by binding to its 5
end. In this respect, it is worth noting that Pol ␮ still contains
an 8 kDa domain though it does not have a dRP lyase activity,
and that this domain, like that of Pol ␭, recognizes a phosphate
at the 5 end of the gap [83].
The promiscuity with which Pol ␭ generates deletion errors is
surprising in a DNA polymerase with an otherwise moderately
high fidelity of synthesis and supposedly involved in DNA repair.
However, this characteristic suggests that Pol ␭ has adapted to be
able to bind substrates with minimal homology. This is one of the
requirements that a DNA polymerase should have in order to par-
ticipate in the NHEJ process, where the initial complementarity
between the strands to be joined can be low or even nonexis-
tent. Interestingly, the two other family X enzymes involved in
NHEJ, Pol IV [36] and Pol ␮ [84], also have a very low fidelity
for deletions, while Pol ␤, mainly involved in BER, is much
more accurate [85].
Finally, recent biochemical [81] data suggest that subtle struc-
tural differences among family X DNA polymerases might be
responsible for the nature of the DNA ends that they can act
1365
upon. For example, the ability of different family X enzymes
to conduct gap-filling in a NHEJ context is dependent on the
identity of a loop in their C-terminus. This loop is longer for
Pol ␮ than for Pol ␭, and seems to provide the former with
some ability to conduct template-independent synthesis (Juarez-
Santos and Blanco, in preparation). Moreover, it enables Pol ␮
to conduct gap-filling on a substrate that is missing the template
nucleotide corresponding to the terminal base pair. The inability
of Pol ␭ to conduct synthesis on such a substrate is consistent
with biochemical and structural data indicating that it requires a
correct terminal base pair for optimal synthesis efficiency. This
significant difference between Pol ␭ and Pol ␮ suggests that
the intrinsic abilities of the different DNA polymerases might
define which polymerase may participate in each end-joining
transaction.
Acknowledgements
The authors wish to thank Drs. Scott McCulloch and Zachary
Pursell for critical reading of the manuscript. This research was
supported in part by the Intramural Research Program of the
NIH, and NIEHS. Dale Mosbaugh was an outstanding nucleic
acid biochemist who devoted considerable efforts to characteriz-
ing DNA polymerase ␤ [86–91], the prototype family X enzyme.
Almost 25 years after his seminal early work on Pol ␤, it is a
bittersweet pleasure to dedicate this brief review of a family X
enzyme to his memory.
References
[1] J.H. Hoeijmakers, Genome maintenance mechanisms for preventing can-
cer, Nature 411 (2001) 366–374.
[2] K. Bebenek, T.A. Kunkel, Functions of DNA polymerases, in: DNA
Repair and Replication, Elsevier Press, San Diego, 2004.
[3] J. Ito, D.K. Braithwaite, Compilation and alignment of DNA polymerase
sequences, Nucleic Acids Res. 19 (1991) 4045–4057.
[4] D.K. Braithwaite, J. Ito, Compilation, alignment, and phylogenetic rela-
tionships of DNA polymerases, Nucleic Acids Res. 21 (1993) 787–802.
[5] R.W. Sobol, J.K. Horton, R. Kuhn, H. Gu, R.K. Singhal, R. Prasad, K.
Rajewsky, S.H. Wilson, Requirement of mammalian DNA polymerase-
beta in base-excision repair, Nature 379 (1996) 183–186.
[6] R.W. Sobol, S.H. Wilson, Mammalian DNA beta-polymerase in base
excision repair of alkylation damage, Prog. Nucleic Acid Res. Mol.
Biol. 68 (2001) 57–74.
[7] P. Fortini, B. Pascucci, E. Parlanti, M. D’Errico, V. Simonelli, E.
Dogliotti, The base excision repair: mechanisms and its relevance for
cancer susceptibility, Biochimie 85 (2003) 1053–1071.
[8] S. Gilfillan, C. Benoist, D. Mathis, Mice lacking terminal deoxynu-
cleotidyl transferase: adult mice with a fetal antigen receptor repertoire,
Immunol. Rev. 148 (1995) 201–219.
[9] K. Shimizu, C. Santocanale, P.A. Ropp, M.P. Longhese, P. Plevani, G.
Lucchini, A. Sugino, Purification and characterization of a new DNA
polymerase from budding yeast Saccharomyces cerevisiae. A probable
homolog of mammalian DNA polymerase beta, J. Biol. Chem. 268
(1993) 27148–27153.
[10] R. Prasad, S.G. Widen, R.K. Singhal, J. Watkins, L. Prakash, S.H.
Wilson, Yeast open reading frame YCR14C encodes a DNA beta-
polymerase-like enzyme, Nucleic Acids Res. 21 (1993) 5301–5307.
[11] P. Bork, K. Hofmann, P. Bucher, A.F. Neuwald, S.F. Altschul, E.V.
Koonin, A superfamily of conserved domains in DNA damage-
responsive cell cycle checkpoint proteins, FASEB J. 11 (1997) 68–76.
1366 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367
[12] I. Callebaut, J.P. Mornon, From BRCA1 to RAP1: a widespread BRCT
module closely associated with DNA repair, FEBS Lett. 400 (1997)
25–30.
[13] S.H. Leem, P.A. Ropp, A. Sugino, The yeast Saccharomyces cerevisiae
DNA polymerase IV: possible involvement in double strand break DNA
repair, Nucleic Acids Res. 22 (1994) 3011–3017.
[14] M. Mclnnis, G. O’Neill, K. Fossum, M.S. Reagan, Epistatic analysis of
the roles of the RAD27 and POL4 gene products in DNA base excision
repair in S. cerevisiae, DNA Repair (Amst.) 1 (2002) 311–315.
[15] T.E. Wilson, M.R. Lieber, Efficient processing of DNA ends during
yeast nonhomologous end joining. Evidence for a DNA polymerase beta
(Pol4)-dependent pathway, J. Biol. Chem. 274 (1999) 23599–23609.
[16] M. Garcia-Diaz, O. Dominguez, L.A. Lopez-Fernandez, L.T. de Lera,
M.L. Saniger, J.F. Ruiz, M. Parraga, M.J. Garcia-Ortiz, T. Kirchhoff, J.
del Mazo, A. Bernad, L. Blanco, DNA polymerase lambda (Pol lambda),
a novel eukaryotic DNA polymerase with a potential role in meiosis, J.
Mol. Biol. 301 (2000) 851–867.
[17] S. Aoufouchi, E. Flatter, A. Dahan, A. Faili, B. Bertocci, S. Storck,
F. Delbos, L. Cocea, N. Gupta, J.C. Weill, C.A. Reynaud, Two novel
human and mouse DNA polymerases of the polX family, Nucleic Acids
Res. 28 (2000) 3684–3693.
[18] K. Nagasawa, K. Kitamura, A. Yasui, Y. Nimura, K. Ikeda, M. Hirai, A.
Matsukage, M. Nakanishi, Identification and characterization of human
DNA polymerase beta 2, a DNA polymerase beta-related enzyme, J.
Biol. Chem. 275 (2000) 31233–31238.
[19] O. Dominguez, J.F. Ruiz, T. Lain de Lera, M. Garcia-Diaz, M.A. Gonza-
lez, T. Kirchhoff, A.C. Martinez, A. Bernad, L. Blanco, DNA polymerase
mu (Pol mu), homologous to TdT, could act as a DNA mutator in
eukaryotic cells, EMBO J. 19 (2000) 1731–1742.
[20] Y. Uchiyama, S. Kimura, T. Yamamoto, T. Ishibashi, K. Sakaguchi,
Plant DNA polymerase lambda, a DNA repair enzyme that functions
in plant meristematic and meiotic tissues, Eur. J. Biochem. 271 (2004)
2799–2807.
[21] H. Ohmori, E.C. Friedberg, R.P. Fuchs, M.F. Goodman, F. Hanaoka, D.
Hinkle, T.A. Kunkel, C.W. Lawrence, Z. Livneh, T. Nohmi, L. Prakash,
S. Prakash, T. Todo, G.C. Walker, Z. Wang, R. Woodgate, The Y-family
of DNA polymerases, Mol. Cell 8 (2001) 7–8.
[22] W. Yang, Damage repair DNA polymerases Y, Curr. Opin. Struct. Biol.
13 (2003) 23–30.
[23] M.F. Goodman, Error-prone repair DNA polymerases in prokaryotes and
eukaryotes, Annu. Rev. Biochem. 71 (2002) 17–50.
[24] I.K. Cann, Y. Ishino, Archaeal DNA replication: identifying the pieces
to solve a puzzle, Genetics 152 (1999) 1249–1267.
[25] M. Garcia-Diaz, K. Bebenek, R. Sabariegos, O. Dominguez, J.
Rodriguez, T. Kirchhoff, E. Garcia-Palomero, A.J. Picher, R. Juarez, J.F.
Ruiz, T.A. Kunkel, L. Blanco, DNA polymerase lambda, a novel DNA
repair enzyme in human cells, J. Biol. Chem. 277 (2002) 13184–13191.
[26] T.A. Steitz, DNA polymerases: structural diversity and common mech-
anisms, J. Biol. Chem. 274 (1999) 17395–17398.
[27] M. Garcia-Diaz, K. Bebenek, J.M. Krahn, L. Blanco, T.A. Kunkel,
L.C. Pedersen, A structural solution for the DNA polymerase lambda-
dependent repair of DNA gaps with minimal homology, Mol. Cell 13
(2004) 561–572.
[28] N. Shimazaki, K. Yoshida, T. Kobayashi, S. Toji, K. Tamai, O. Koiwai,
Over-expression of human DNA polymerase lambda in E. coli and char-
acterization of the recombinant enzyme, Genes Cells 7 (2002) 639–651.
[29] K. Ramadan, G. Maga, I.V. Shevelev, G. Villani, L. Blanco, U. Hub-
scher, Human DNA polymerase lambda possesses terminal deoxyribonu-
cleotidyl transferase activity and can elongate RNA primers: implications
for novel functions, J. Mol. Biol. 328 (2003) 63–72.
[30] I. Shevelev, G. Blanca, G. Villani, K. Ramadan, S. Spadari, U. Hubscher,
G. Maga, Mutagenesis of human DNA polymerase lambda: essential
roles of Tyr505 and Phe506 for both DNA polymerase and terminal
transferase activities, Nucleic Acids Res. 31 (2003) 6916–6925.
[31] H. Pelletier, M.R. Sawaya, W. Woffle, S.H. Wilson, J. Kraut, Crystal
structures of human DNA polymerase beta complexed with DNA: impli-
cations for catalytic mechanism, processivity, and fidelity, Biochemistry
35 (1996) 12742–12761.
[32] J.M. Clark, Novel non-templated nucleotide addition reactions catalyzed
by procaryotic and eucaryotic DNA polymerases, Nucleic Acids Res.
16 (1988) 9677–9686.
[33] G. Maga, G. Villani, K. Ramadan, I. Shevelev, N. Tanguy Le Gac,
L. Blanco, G. Blanca, S. Spadari, U. Hubscher, Human DNA poly-
merase lambda functionally and physically interacts with proliferating
cell nuclear antigen in normal and translesion DNA synthesis, J. Biol.
Chem. 277 (2002) 48434–48440.
[34] N. Shimazaki, T. Yazaki, T. Kubota, A. Sato, A. Nakamura, S. Kurei, S.
Toji, K. Tamai, O. Koiwai, DNA polymerase lambda directly binds to
proliferating cell nuclear antigen through its confined C-terminal region,
Genes Cells 10 (2005) 705–715.
[35] K. Bebenek, M. Garcia-Diaz, L. Blanco, T.A. Kunkel, The frameshift
infidelity of human DNA polymerase lambda: implications for function,
J. Biol. Chem. 278 (2003) 34685–34690.
[36] K. Bebenek, M. Garcia-Diaz, S.R. Patishall, T.A. Kunkel, Biochemical
properties of Saccharomyces cerevisiae DNA polymerase IV, J. Biol.
Chem. 280 (2005) 20051–20058.
[37] H.E. Krokan, H. Nilsen, F. Skorpen, M. Otterlei, G. Slupphaug, Base
excision repair of DNA in mammalian cells, FEBS Lett. 476 (2000)
73–77.
[38] M. Garcia-Diaz, K. Bebenek, T.A. Kunkel, L. Blanco, Identification of
an intrinsic 5 -deoxyribose-5-phosphate lyase activity in human DNA
polymerase lambda: a possible role in base excision repair, J. Biol.
Chem. 276 (2001) 34659–34663.
[39] E.K. Braithwaite, R. Prasad, D.D. Shock, E.W. Hou, W.A. Beard, S.H.
Wilson, DNA polymerase lambda mediates a back-up base excision
repair activity in extracts of mouse embryonic fibroblasts, J. Biol. Chem.
280 (2005) 18469–18475.
[40] E.K. Braithwaite, P.S. Kedar, L. Lan, Y.Y. Polosina, K. Asagoshi, V.P.
Poltoratsky, J.K. Horton, H. Miller, G.W. Teebor, A. Yasui, S.H. Wilson,
DNA polymerase lambda protects mouse fibroblasts against oxidative
DNA damage and is recruited to sites of DNA damage/repair, J. Biol.
Chem. (2005).
[41] W.A. Beard, S.H. Wilson, Structural design of a eukaryotic DNA repair
polymerase: DNA polymerase beta, Mutat. Res. 460 (2000) 231–244.
[42] R.W. Sobol, R. Prasad, A. Evenski, A. Baker, X.P. Yang, J.K. Hor-
ton, S.H. Wilson, The lyase activity of the DNA repair protein beta-
polymerase protects from DNA-damage-induced cytotoxicity, Nature
405 (2000) 807–810.
[43] S.L. Allinson, Dianova II, G.L. Dianov, DNA polymerase beta is the
major dRP lyase involved in repair of oxidative base lesions in DNA
by mammalian cell extracts, EMBO J. 20 (2001) 6919–6926.
[44] H.T. Idriss, O. Al-Assar, S.H. Wilson, DNA polymerase beta, Int. J.
Biochem. Cell. Biol. 34 (2002) 321–324.
[45] C.E. Piersen, A.K. McCullough, R.S. Lloyd, AP lyases and dRPases:
commonality of mechanism, Mutat. Res. 459 (2000) 43–53.
[46] Y. Matsumoto, K. Kim, Excision of deoxyribose phosphate residues by
DNA polymerase beta during DNA repair, Science 269 (1995) 699–702.
[47] A.J. Kurtz, M.L. Dodson, R.S. Lloyd, Evidence for multiple imino inter-
mediates and identification of reactive nucleophiles in peptide-catalyzed
beta-elimination at abasic sites, Biochemistry 41 (2002) 7054–7064.
[48] Y. Matsumoto, K. Kim, D.S. Katz, J.A. Feng, Catalytic center of DNA
polymerase beta for excision of deoxyribose phosphate groups, Bio-
chemistry 37 (1998) 6456–6464.
[49] L.J. Deterding, R. Prasad, G.P. Mullen, S.H. Wilson, K.B. Tomer, Map-
ping of the 5 -2-deoxyribose-5-phosphate lyase active site in DNA
polymerase beta by mass spectrometry, J. Biol. Chem. 275 (2000)
10463–10471.
[50] E.F. DeRose, T.W. Kirby, G.A. Mueller, K. Bebenek, M. Garcia-Diaz,
L. Blanco, T.A. Kunkel, R.E. London, Solution structure of the lyase
domain of human DNA polymerase lambda, Biochemistry 42 (2003)
9564–9574.
[51] J.A. Wilde, P.H. Bolton, A. Mazumder, M. Manoharan, J.A. Gerlt, Char-
acterization of the equilibrating forms of the aldehydic abasic site in
duplex DNA by O-17 NMR, J. Am. Chem. Soc. 111 (1989) 1894–1896.
[52] G.P. Mullen, W. Antuch, M.W. Maciejewski, R. Prasad, S.H. Wil-
son, Insights into the mechanism of the b-elimination catalyzed by
 M. Garcia-Diaz et al. / DNA Repair 4 (2005) 1358–1367
the N-terminal domain of DNA polymerase b, Tetrahedron 53 (1997)
12057–12066.
[53] J.A. Feng, C.J. Crasto, Y. Matsumoto, Deoxyribose phosphate excision
by the N-terminal domain of the polymerase beta: the mechanism revis-
ited, Biochemistry 37 (1998) 9605–9611.
[54] R. Prasad, W.A. Beard, J.Y. Chyan, M.W. Maciejewski, G.P. Mullen,
S.H. Wilson, Functional analysis of the amino-terminal 8-kDa domain
of DNA polymerase beta as revealed by site-directed mutagenesis. DNA
binding and 5 -deoxyribose phosphate lyase activities, J. Biol. Chem.
273 (1998) 11121–11126.
[55] O.M. Sidorkina, J. Laval, Role of the N-terminal pro line residue in the
catalytic activities of the Escherichia coli Fpg protein, J. Biol. Chem.
275 (2000) 9924–9929.
[56] T.T. Saxowsky, Y. Matsumoto, P.T. Englund, The mitochondrial DNA
polymerase beta from Crithidia fasciculata has 5 -deoxyribose phosphate
(dRP) lyase activity but is deficient in the release of dRP, J. Biol. Chem.
277 (2002) 37201–37206.
[57] D. Wong, B. Demple, Modulation of the 5 -deoxyribose-5-phosphate
lyase and DNA synthesis activities of mammalian DNA polymerase
beta by apurinic/apyrimidinic endonuclease 1, J. Biol. Chem. 279 (2004)
25268–25275.
[58] M.R. Sawaya, R. Prasad, S.H. Wilson, J. Kraut, H. Pelletier, Crystal
structures of human DNA polymerase beta complexed with gapped and
nicked DNA: evidence for an induced fit mechanism, Biochemistry 36
(1997) 11205–11215.
[59] S. Doublie, M.R. Sawaya, T. Ellenberger, An open and closed case for
all polymerases, Structure 7 (1999) R31–R355.
[60] Y. Li, S. Korolev, G. Waksman, Crystal structures of open and closed
forms of binary and ternary complexes of the large fragment of Thermus
aquaticus DNA polymerase I: structural basis for nucleotide incorpora-
tion, EMBO J. 17 (1998) 7514–7525.
[61] T.A. Kunkel, K. Bebenek, DNA replication fidelity, Annu. Rev. Biochem.
69 (2000) 497–529.
[62] T.A. Kunkel, DNA replication fidelity, J. Biol. Chem. 279 (2004)
16895–16898.
[63] C.M. Joyce, S.J. Benkovic, DNA polymerase fidelity: kinetics, structure,
and checkpoints, Biochemistry 43 (2004) 14317–14324.
[64] M. Garcia-Diaz, K. Bebenek, J.M. Krahn, T.A. Kunkel, L.C. Pedersen,
A closed conformation for the Pol lambda catalytic cycle, Nat. Struct.
Mol. Biol. (2005).
[65] S. Doublie, T. Ellenberger, The mechanism of action of T7 DNA poly-
merase, Curr. Opin. Struct. Biol. 8 (1998) 704–712.
[66] S.J. Johnson, J.S. Taylor, L.S. Beese, Processive DNA synthesis observed
in a polymerase crystal suggests a mechanism for the prevention
of frameshift mutations, Proc. Natl. Acad. Sci. U.S.A. 100 (2003)
3895–3900.
[67] M.C. Franklin, J. Wang, T.A. Steitz, Structure of the replicating complex
of a pol alpha family DNA polymerase, Cell 105 (2001) 657–667.
[68] T.A. Baker, A. Kornberg, DNA Replication, W.H. Freeman and Com-
pany, New York, 1992.
[69] F. Eckstein, J.B. Thomson, Phosphate analogs for study of DNA poly-
merases, Methods Enzymol. 262 (1995) 189–202.
[70] P.M. Burgers, F. Eckstein, A study of the mechanism of DNA poly-
merase I from Escherichia coli with diastereomeric phosphorothioate
analogs of deoxyadenosine triphosphate, J. Biol. Chem. 254 (1979)
6889–6893.
[71] T.A. Steitz, S.J. Smerdon, J. Jager, C.M. Joyce, A unified polymerase
mechanism for nonhomologous DNA and RNA polymerases, Science
266 (1994) 2022–2025.
[72] J.W. Lee, L. Blanco, T. Zhou, M. Garcia-Diaz, K. Bebenek, T.A.
Kunkel, Z. Wang, L.F. Povirk, Implication of DNA polymerase lambda
in alignment-based gap filling for nonhomologous DNA end joining in
human nuclear extracts, J. Biol. Chem. 279 (2003) 805–811.
1367
[73] W. Fan, X. Wu, DNA polymerase lambda can elongate on DNA
substrates mimicking non-homologous end joining and interact with
XRCC4-ligase IV complex, Biochem. Biophys. Res. Commun. 323
(2004) 1328–1333.
[74] Y. Ma, H. Lu, B. Tippin, M.F. Goodman, N. Shimazaki, O. Koiwai,
C.L. Hsieh, K. Schwarz, M.R. Lieber, A biochemically defined system
for mammalian nonhomologous DNA end joining, Mol. Cell 16 (2004)
701–713.
[75] K. Valerie, L.F. Povirk, Regulation and mechanisms of mammalian
double-strand break repair, Oncogene 22 (2003) 5792–5812.
[76] M.R. Lieber, Y. Ma, U. Pannicke, K. Schwarz, Mechanism and regula-
tion of human non-homologous DNA end-joining, Nat. Rev. Mol. Cell.
Biol. 4 (2003) 712–720.
[77] S.A. Nick McElhinny, D.A. Ramsden, Sibling rivalry: competition
between Pol X family members in V(D)J recombination and general
double strand break repair, Immunol. Rev. 200 (2004) 156–164.
[78] M.R. Lieber, Y. Ma, U. Pannicke, K. Schwarz, The mechanism of
vertebrate nonhomologous DNA end joining and its role in V(D)J recom-
bination, DNA Repair (Amst.) 3 (2004) 817–826.
[79] K.N. Mahajan, S.A. Nick McElhinny, B.S. Mitchell, D.A. Ramsden,
Association of DNA polymerase mu (pol mu) with Ku and ligase IV:
role for pol mu in end-joining double-strand break repair, Mol. Cell.
Biol. 22 (2002) 5194–5202.
[80] H.M. Tseng, A.E. Tomkinson, A physical and functional interaction
between yeast Pol4 and Dnl4-Lif1 links DNA synthesis and ligation in
nonhomologous end joining, J. Biol. Chem. 277 (2002) 45630–45637.
[81] S.A. Nick McElhinny, J.M. Havener, M. Garcia-Diaz, R. Juarez, K.
Bebenek, B.L. Kee, L. Blanco, T.A. Kunkel, D.A. Ramsden, A gradi-
ent of template dependence defines distinct biological roles for family
x polymerases in nonhomologous end joining, Mol. Cell 19 (2005)
357–366.
[82] B. Bertocci, A. De Smet, C. Berek, J.C. Weill, C.A. Reynaud,
Immunoglobulin kappa light chain gene rearrangement is impaired in
mice deficient for DNA polymerase mu, Immunity 19 (2003) 203–
211.
[83] S.A. Nick McElhinny, D.A. Ramsden, Polymerase mu is a DNA-directed
DNA/RNA polymerase, Mol. Cell. Biol. 23 (2003) 2309–2315.
[84] Y. Zhang, X. Wu, F. Yuan, Z. Xie, Z. Wang, Highly frequent frameshift
DNA synthesis by human DNA polymerase mu, Mol. Cell. Biol. 21
(2001) 7995–8006.
[85] T.A. Kunkel, Frameshift mutagenesis by eucaryotic DNA polymerases
in vitro, J. Biol. Chem. 261 (1986) 13581–13587.
[86] G.S. Probst, D.M. Stalker, D.W. Mosbaugh, R.R. Meyer, Stimulation
of DNA polymerase by factors isolated from Novikoff hepatoma, Proc.
Natl. Acad. Sci. U.S.A. 72 (1975) 1171–1174.
[87] D.W. Mosbaugh, T.A. Kunkel, D.M. Stalker, J.E. Tcheng, R.R. Meyer,
Novikoff hepatoma deoxyribonucleic acid polymerase. Sensitivity of the
beta-polymerase to sulfhydryl blocking agents, Nucleic Acids Res. 3
(1976) 2341–2352.
[88] D.M. Stalker, D.W. Mosbaugh, R.R. Meyer, Novikoff hepatoma deoxyri-
bonucleic acid polymerase. Purification and properties of a homogeneous
beta polymerase, Biochemistry 15 (1976) 3114–3121.
[89] D.W. Mosbaugh, D.M. Stalker, G.S. Probst, R.R. Meyer, Novikoff
hepatoma deoxyribonucleic acid polymerase. Identification of a stim-
ulatory protein bound to the beta-polymerase, Biochemistry 16 (1977)
1512–1518.
[90] D.W. Mosbaugh, R.R. Meyer, Interaction of mammalian deoxyribonu-
clease V, a double strand 3 to 5 and 5 to 3 exonuclease, with
deoxyribonucleic acid polymerase-beta from the Novikoff hepatoma, J.
Biol. Chem. 255 (1980) 10239–10247.
[91] D.W. Mosbaugh, S. Linn, Excision repair and DNA synthesis with a
combination of HeLa DNA polymerase beta and DNase V, J. Biol.
Chem. 258 (1983) 108–118.