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Function of Translesion DNA polymerases in Genome Stability, 2015: 73-90
ISBN: 978-81-308-0538-2 Editors: Domenico Maiorano & Jean-Sébastien Hoffmann
5. DNA polymerase eta
Chikahide Masutani1, Rie Kanao1 and Fumio Hanaoka2
1Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan; 2Faculty of Science, Gakushuin
University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan
Abstract. DNA polymerase (Pol ) is the only one of the fifteen
human DNA-dependent DNA polymerases for which a natural
occurring deficiency is known to predispose humans to cancer.
Xeroderma pigmentosum (XP) is an autosomal recessive genetic
disorder, associated with a greatly increased risk of sunlight-
induced skin tumors, and individuals with the variant type of this
syndrome, XP-V, have defects in Pol . Pol has a DNA
polymerase activity capable of catalyzing translesion DNA
synthesis past the most prominent UV-induced lesion, a cis-syn
TT-cyclobutane pyrimidine dimer (CPD), with high efficiency and
fidelity. Crystal structure of human Pol complexed with CPD-
containing template-primer DNA reveals that Pol is the DNA
polymerase for bypassing CPD lesion. In addition, mammalian
Pol has other physiological functions including somatic
hypermutation, homologous recombination and replication of
common fragile sites. Pol recruitment is regulated by mono-
ubiquitination and de-ubiquitination of PCNA at least in part.
Correspondence/Reprint request: Dr. Fumio Hanaoka, Faculty of Science, Gakushuin University, 1-5-1 Mejiro,
Toshima-ku, Tokyo 171-8588, Japan. E-mail: [email protected]
Chikahide Masutani et al. 74
Introduction
In 1999, the protein encoded by the RAD30 gene of Saccharomyces
cerevisiae was shown to have an intrinsic DNA polymerase activity capable
of catalyzing translesion DNA synthesis (TLS) past the most prominent type
of UV-induced lesion, the cyclobutane pyrimidine dimer (CPD) [1]. Rad30
was the seventh DNA template–dependent DNA polymerase identified in
eukaryotes, and it was named DNA polymerase (Pol ). In the same year,
using an in vitro DNA replication system with a CPD-containing DNA
template, we identified a human protein that corrects the defect of cell-free
extracts from cells of xeroderma pigmentosum variant (XP-V) and has a
DNA polymerase activity capable of bypassing CPD lesions [2]. The gene
that encodes the latter protein turned out to be a human homologue of yeast
RAD30 [3]; it was initially named XPV/RAD30A, and is now called POLH
following unification of the nomenclature. Independently, the same gene was
isolated by homology to yeast RAD30 [4]. Mutations in the POLH gene have
been identified in XP-V patients [3,4,5,6], and the wild-type gene has the
ability to correct the UV sensitivity of XP-V cells [7,8]. At roughly the same
time, another translesion DNA polymerase, Pol was also identified as a
mammalian homologue of the yeast Rad30 protein [9]. The mammalian gene
was originally named RAD30B, but following unification it is now called POLI.
Figure 1. Prominent contributions of nucleotide excision repair and TLS to
UV-induced DNA lesions. 6-4PP: 6-4 pyrimidine-pyrimidone photoproduct.
CPD: cyclobutane pyrimidine dimer.
DNA polymerase eta 75
However, unlike the case of POLH, no mutation in POLI has been identified
in XP-V patients.
Xeroderma pigmentosum (XP) is an autosomal recessive genetic
disorder characterized by sunlight sensitivity, cutaneous and ocular
deterioration, premature malignant skin neoplasms, and an increased
incidence of skin cancer after sunlight exposure. XP has been classified into
eight complementation groups: XP-A to XP-G and XP-V. The proteins
deficient in XP-A to XP-G play crucial roles in removal of DNA lesions by
nucleotide excision repair (NER) and maintenance of genome integrity. The
protein that is defective in XP-V, Pol , also plays an important role in
maintaining genome integrity, but in this case by catalyzing translesion
synthesis (TLS) of damaged DNA (Figure 1).
In vivo functions
As in human patients, Pol -deficient mice are viable, fertile, and do not
exhibit any apparent spontaneous physiological defects under normal
conditions. Also similar to humans, fibroblasts from these mice exhibit
enhanced sensitivity to UV, and all Pol -deficient mice developed skin
tumors following UV irradiation. These results are consistent with the
observation that Pol prevents UV-induced cell death and skin cancer by
catalyzing the accurate bypass of CPDs in vivo [10,11]. In addition to UV
irradiation, XP-V cells also exhibit sensitivity to cisplatin [12].
In addition to its role in the tolerance of environmentally induced DNA
lesions, human Pol is also required for common fragile-site stability during
unperturbed DNA replication [13,14]. Because rearrangements of common
fragile sites are a driving force of oncogenesis, this activity of Pol likely
makes a major contribution to the maintenance of genome integrity. On the
other hand, Pol also has mutagenic functions. In peripheral blood
lymphocytes from XP-V patients, the rate of A:T mutation in the
immunoglobulin variable gene is reduced, although the overall mutation
frequency is normal [15,16,17,18]. Consistent results have been observed in
Pol -deficient mice [19,20]. It has been proposed that multiple DNA
polymerases, including Pol , Pol , and REV1, participate in somatic
hypermutation, and that Pol is the main mutator at A and T residues [21].
Furthermore, human Pol has the potential to contribute to mutagenesis at
sites of oxidative DNA damage induced by azathioprine and UV-A light
[22].
In chicken DT40 cells, disruption of Pol causes a decrease in the
frequency of immunoglobulin gene conversion and double-strand
Chikahide Masutani et al. 76
break–induced homologous recombination [23]. Furthermore, human Pol
has the ability to synthesize DNA from D-loop recombination intermediates
in vitro, and this activity is stimulated by interaction with the recombination
protein Rad51 [24]. Together, these data suggest that Pol may also play an
important role in homologous recombination.
Enzymatic properties
Pol is a member of the Y-family of DNA polymerases, which includes
the human enzymes Pol , Pol , and REV1 [25]. Y-family DNA polymerases
are characterized by low processivity and a lack of exonucleolytic
proofreading activity, which makes them error-prone but also able to
catalyze TLS past certain DNA lesions. In fact, in both humans and yeast,
Pol generate base substitutions with error rates of 10-2
to 10-3
when
replicating undamaged DNA. The most striking feature of Pol is its ability
to catalyze TLS past CPD lesions as efficiently as replication of undamaged
DNA [26,27,28,29,30,31]. Importantly, Pol has the ability to preferentially
incorporate adenines opposite damaged thymines of cis-syn TT-CPDs,
although misincorporations may sometimes take place [27,28]. Intriguingly,
Pol preferentially binds to template-primer DNAs consisting of TT-CPD
templates and primers whose 3’ ends are the correctly paired nucleotides
situated opposite the TT of the CPD and the immediately following
nucleotide. This allows Pol to replicate past TT-CPDs without dissociating
from the template primer when the correct nucleotides are incorporated. On
the other hand, if the incorrect nucleotides are incorporated, Pol readily
dissociates from the template primer. As a result, the replicated DNA
contains the correct nucleotides opposite the lesion; thus, Pol bypasses
CPDs with biased fidelity (Figure 2) [32,33]. On the other hand, Pol cannot
bypass another major UV-induced lesion, the 6-4 photoproduct, without
assistance from other enzymes. Conversely, NER, which is missing in cells
from XP-A to XP-G patients, removes 6-4 photoproducts efficiently and
prevents skin cancer, but removes CPDs inefficiently throughout the genome
[34]. Thus, TLS and NER represent complementary systems, both of which
are important for UV damage tolerance in humans (Figure 1).
Consistent with the cisplatin sensitivity of XP-V cells [12], Pol can
bypass cisplatin adducts in vitro [35] and, together with Pol , catalyze TLS
past cisplatin adducts both in human cells [36] and in vitro [37]. Pol also
contributes to TLS across lesions, such as 8-oxoguanine, thymine glycols,
acetylaminofluorene adducts, and BPDE adducts [28,31,36,38,39]. However,
in contrast to bypass of CPDs, in which Pol acts as the main polymerase,
DNA polymerase eta 77
Figure 2. A model for DNA polymerase switching during TLS, reproduced from
Kusumoto et al. 2004 [32]. A replicative DNA polymerase stalls at a TT dimer. (1)
Pol binds the template/primer at the site of the TT dimer and (2) preferentially
incorporates dAMP opposite the 3’ T of the TT dimer. (3) The association of Pol
with the template/primer DNA becomes more stable. Consequently, Pol is able to
incorporate a nucleotide opposite the 5’ T of the TT dimer. (4) After Pol
incorporates two more nucleotides beyond the TT dimer, the association of Pol with
the DNA becomes unstable, and Pol dissociates. (5) The replication polymerase can
resume DNA synthesis. (6) If Pol incorporates dCMP, dGMP, or dTMP opposite
the 3’ T of the TT dimer (7), it’s binding to the template/primer DNA is not
stabilized. (8) Exonuclease activity excises the incorrect nucleotide opposite the 3’ T
of the TT dimer, allowing Pol to attack the template/primer substrate again.
Pol may contribute to bypass of non-CPD lesions as just one of several enzymes, and it may cause errors in this context [40]. Thus, depending on the lesion, cells may use a variety of mechanisms to select the appropriate polymerase.
Molecular structure
Although sequences conserved among replicative DNA polymerases are
not present in Y-family polymerases, structural analyses revealed that the
Chikahide Masutani et al. 78
overall topologies of the Y-family catalytic domains are similar to those of
replicative polymerases. This topology can be likened to a right hand with
palm, finger, and thumb domains (Figure 3). However, members of the Y
family have several unique characteristics [41,42,43]. First, Y-family
polymerases have spacious active sites that can facilitate lesion bypass to
compensate for their low fidelity. The structure of the palm domain is highly
conserved between Y-family and replicative DNA polymerases, although the
finger and thumb domains of Y-family polymerases are smaller and stubbier.
Second, Y-family polymerases have a unique domain called the ‘little finger’
or polymerase-associated domain (PAD). These structures weaken the
interactions between polymerases, DNA, and incoming nucleotides,
contributing to the relatively lower processivity and poorer fidelity of
Y-family polymerases. Importantly, however, human Pol has a specialized
structure for CPD bypass that acts as a ‘molecular splint’ to stabilize
damaged DNA in a normal conformation during DNA synthesis through the
CPD [44]. Yeast Pol also has a catalytic domain capable of catalyzing
efficient and accurate bypass of CPD lesions [45]. The crystal structures of
Figure 3. Schematic representation of domain structure of human Pol . A.
Secondary structure. B. Steric structure of the ternary complex with DNA,
reproduced from Biertümpfel et al. 2010 [44]. The catalytic domain consists of palm,
thumb, finger, and little finger/PAD domains. The C-terminal region contains
regulatory elements. PIP: PCNA-interacting protein box. RIR: REV1-interacting
region. UBZ: ubiquitin-binding zinc finger. NLS: nuclear localization signal.
DNA polymerase eta 79
human Pol bypassing a cisplatin-induced intrastrand crosslink [46,47] and
yeast Pol bypassing an 8-oxoG lesion [48] have been also solved.
Intriguingly, catalysis of the formation of the phosphodiester bond by human
Pol has been visualized by time-resolved X-ray crystallography [49],
making it possible to reveal more detailed reaction mechanisms in the future.
Regulation by mono-ubiquitinated PCNA
The N-terminal half of Pol contains residues conserved among Y-
family DNA polymerases. Human Pol consists of 713 amino acids, but the
originally identified protein that corrected replication defects of XP-V cell-
free extracts contained only the N-terminal 511 amino acids, but still had
DNA polymerase activity [2,3]. Consistent with this, the N-terminal 432
residues of Pol exhibit basal DNA polymerase activity, but cannot correct
the UV sensitivity of XP-V cells [50]. Together, these observations indicate
that the C-terminal residues are dispensable for DNA polymerase activity,
but necessary for the protein’s proper function in cells (Figure 3A).
Overproduction of Pol does not raise the rate of spontaneous mutation in
human cells despite its intrinsically mutagenic properties, suggesting that its
mutagenic activity is subject to tight regulation [51].
The nuclear localization signal and PCNA interaction peptide (PIP)
sequences, located close to the C-terminus, are important for the cellular
localization of Pol and the formation of nuclear foci with PCNA after UV
irradiation, respectively [8]. Although foci formation by Pol is almost
completely abolished by mutations in the PIP sequences, the ability of such
Pol mutants to rescue the UV sensitivity of XP-V cells is only partially
compromised. Thus, nuclear foci formation is not always required for TLS
by Pol . An ubiquitin-binding zinc finger (UBZ) domain is required for the
interaction of Pol with monoubiquitinated PCNA, as well as for its
relocalization to damaged chromatin [52,53]. Several point mutations in the
UBZ domain severely affect the ability of XP-V cells to cope with
UV-induced DNA damage. Another PIP-like domain is located upstream of
the UBZ domain [54], and an allele of Pol lacking the C-terminus
(including this UBZ sequence) but retaining an intact PIP-like motif can
promote cellular survival in response to UV [50]. Thus, the roles played by
each of these regions in the functions of Pol remain somewhat unclear. In
addition, cells are able to activate and relocate Pol independently of PCNA
monoubiquitination [55,56,57,58]. Other Y-family polymerases also contain
domains that interact with PCNA and/or ubiquitin; however, among all the
TLS polymerases, only Pol interacts with RAD18, which is the primary
Chikahide Masutani et al. 80
ubiquitin ligase responsible for PCNA monoubiquitination; the
RAD18-Pol interaction is crucial for guiding Pol to arrested replication
forks after UV irradiation [59]. Conversely, Pol enhances recruitment of
RAD18 and mono-ubiquitination of PCNA at stalled replication forks [60].
Thus, there may be positive feedback between PCNA ubiquitination and the
recruitment of Pol and RAD18 proteins. Several other proteins also
influence the ubiquitination of PCNA, including Spartan/C1orf124/DVC1,
NBS1, PTIP/Swift, CHK1-Claspin, and ELG1 [61].
Regulation by posttranslational modifications
Pol undergoes several types of posttranslational modifications. ATR-
or PKC-mediated phosphorylation of Pol at C-terminal residues activates
Pol [62,63]. Phosphorylation of RAD18 by the protein kinase Cdc7 also
regulates the Pol -Rad18 interaction and Pol activation [64]. These
observations suggest an interaction between the DNA damage response and
TLS pathways. Lysines close to the C-terminus of Pol undergo
monoubiquitination, which prevents the interaction between Pol and PCNA
[65]. An E3 ubiquitin ligase, Pirh2, binds to Pol , catalyzes its
monoubiquitination, and suppresses TLS [66]. Thus, monoubiquitination at
the C-terminus may be involved in negative regulation of Pol , e.g., in the
inactivation of Pol after TLS is completed. On the contrary, ubiquitination
of Pol promotes its interaction with Pol [67], suggesting that
ubiquitination is involved in the activation of TLS polymerase switching.
The E3 ubiquitin ligase Mdm2 interacts with Pol and promotes both
polyubiquitination and proteasomal degradation of the polymerase [68]. In
C. elegans, degradation of Pol is mediated by the Cul4-Ddb1-Cdt2
pathway, whereas SUMOylation of Pol counteracts this proteolysis [69].
Thus, proteolytic degradation of Pol and its regulation can control TLS.
The molecular chaperone HSP90 also regulates the stability of Pol in
human cells [70].
Non-canonical roles in TLS regulation
Human Pol interacts with REV1 through two domains located in the
C-terminus (Figure 3A) [71,72]. Mutations in these domains disrupt the
Pol -REV1 interaction, but do not affect the ability of Pol itself to catalyze
TLS past CPDs and promote the survival of XP-V cells following UV
damage [73]. Like Pol , REV1 is a member of the Y-family of DNA
polymerases; in addition, and in conjunction with Pol , it plays a crucial role
DNA polymerase eta 81
in UV-induced mutagenesis. An allele of Pol defective in the ability to
interact with REV1 only partially suppresses spontaneous mutations in XP-V
cells, but suppresses UV-induced mutations completely. REV1 is thought to
play a central role in TLS polymerase switching because it interacts with the
Y-family polymerases Pol and Pol , as well as Pol and Pol through its
C-terminus [71,72,74,75]. The Pol -REV1 interaction stimulates the
accumulation of endogenous REV1 to UV-damaged DNA sites [73], and
Pol promotes Pol foci formation [76], suggesting that cells may initially
preferentially recruit Pol to lesions, but then subsequently switch to other
TLS polymerases. The UV sensitivity of Pol -deficient mouse cells can be
moderately rescued by expression of catalytically inactive Pol but not by
Pol with additional mutations in REV1-interacting motifs [77]. Expression
of the inactive Pol cannot suppress UV-induced mutations in Pol -
deficient cells, but such mutations are ultimately suppressed in Pol -, Pol -,
and Pol -deficient cells; however, Pol has not been demonstrated to
contribute to mutagenesis. Together, these observations suggest that the
Pol -REV1 interaction promotes an alternative TLS pathway in which Pol
plays a mutagenic role (Figure 4). However, because REV1 forms foci in
response to UV irradiation independently of Pol [73,78], and REV1 and
other TLS polymerases interact with monoubiquitinated PCNA directly,
each polymerase could be recruited to arrested replication forks
independently of the others. It is likely that cells have multiple TLS
pathways to choose from, and that they preferentially promote each pathway
under different circumstance.
Figure 4. Model of TLS polymerase selection during TLS in the presence or absence
of Pol . Reproduced and modified from Ito et al. 2012 [77].
Chikahide Masutani et al. 82
Pol also interacts with, and is stimulated by, a PCNA loader complex,
Ctf18-RFC [79]. A Pol -interacting protein, PDIP38, also interacts with
Pol , Rev1, and Pol [80]. Thus, it is likely that the replication and
translesion machinery are connected in various ways to allow cells to
tolerate DNA lesions.
Interaction with other repair proteins
Several DNA repair-related proteins interact with and/or regulate Pol .
Hereditary nonpolyposis colon cancer (HNPCC) is associated with
mutations in mismatch-repair genes, including MLH1, MSH2, and MSH6
[81]. Human Pol interacts with MLH1 and MSH2/MSH6 [82,83]. In
addition to correcting errors during DNA replication, mismatch-repair
proteins also contribute to other mechanisms, including somatic
hypermutation of immunoglobulin genes [84]. These functions are to some
extent separable: for example, MSH2 and MSH6, but not MLH1, participate
in somatic hypermutation [85]. The mutation spectrum of somatic
hypermutation in MSH2- or MSH6-deficient mice, in which mutations at
A:T base pairs in immunoglobulin genes are drastically reduced, is similar to
that of Pol -deficient mice. A:T somatic hypermutation also requires
RAD18 [86] and PCNA monoubiquitination [87], although PCNA
ubiquitination-independent mechanisms have also been considered [58].
MSH2/MSH6 stimulates Pol activity in vitro [82], suggesting that the
interaction between Pol and MSH2/MSH6 may be involved in somatic
hypermutation of immunoglobulin genes. However, MSH2/MSH6, Pol ,
and monoubiquitinated PCNA are also involved in the oxidative stress
response [88]. Thus, the interaction between Pol and mismatch-repair
proteins may be involved in the repair of oxidative stress-induced DNA
lesions.
Fanconi anemia (FA) is characterized by hypopigmentation, bone
marrow failure, developmental defects, and cancer predisposition. At least
15 FANC gene mutations have been identified. Defects in the FANC
pathway result in hypersensitivity to interstrand crosslink (ICL) agents, such
as mitomycin C (MMC). The FANC pathway is involved not only in ICL
repair, but also in several other biological processes. Recently, FANCD2,
one of the proteins responsible for FA, was shown to interact with Pol and
contribute to the recruitment of Pol to sites of damage [89].
Werner syndrome (WRN) is characterized by premature aging and
cancer predisposition [90]. The WRN protein is a RecQ-family DNA
helicase with exonuclease activity. WRN interacts with and stimulates the
DNA polymerase eta 83
polymerase activities of TLS polymerases Pol , Pol , and Pol [91]. WRN
also regulates TLS by regulating RAD18-mediated PCNA ubiquitination via
the WRN-NBS1 interaction [92,93].
Nijmegen breakage syndrome (NBS) is characterized by high sensitivity to ionizing radiation and predisposition to malignancies. Cells from NBS patients exhibit defects in double-stranded DNA break repair and checkpoint controls [94]. Reduction in the level of NBS1 protein sensitizes human cells to UV, indicating that NBS1 is likely to also be involved in the UV damage response [95,96]. Consistent with this idea, NBS1 interacts with and recruits RAD18 to damaged chromatin and regulates Pol -catalyzed TLS past UV-induced lesions [97].
Conclusion and perspectives
Pol plays a crucial role in preventing UV-induced skin cancers, and
deficiency of Pol causes a cancer-prone syndrome. Pol is also involved in
somatic hypermutation of immunoglobulin genes and recombination-related
mechanisms. Considering that Pol is ubiquitously expressed throughout the
body, it may have multiple functions that have not yet been identified.
Posttranslational modifications and protein-protein interactions between TLS
polymerases and other proteins, as well as their regulatory mechanisms, have
been identified. A greater understanding of the physiological relevance and
regulatory mechanisms of Pol in DNA damage tolerance could lead to the
development of new ways to treat cancers and other diseases.
Acknowledgements
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan; by the Mitsubishi Foundation; and by Takeda Science Foundation.
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