Alternative titles; symbols
HGNC Approved Gene Symbol: POLH
Cytogenetic location: 6p21.1 Genomic coordinates (GRCh38) : 6:43,576,185-43,620,523 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 6p21.1 | Xeroderma pigmentosum, variant type | 278750 | Autosomal recessive | 3 |
Using an improved cell-free assay for translesion DNA synthesis, Masutani et al. (1999) isolated a DNA polymerase from HeLa cells that continues replication on damaged DNA by bypassing ultraviolet-induced thymine dimers in xeroderma pigmentosum variant (XPV; 278750) cell extracts. Masutani et al. (1999) demonstrated that this polymerase, symbolized POLH, is a human homolog of the yeast Rad30 protein identified as DNA polymerase eta (Johnson et al., 1999). POLH and yeast Rad30 are members of a family of damage-bypass replication proteins which comprises the Escherichia coli proteins UmuC and DinB and the yeast Rev1 protein. The XPV cDNA encodes a protein of 713 amino acids with a calculated molecular mass of 78.4 kD. The amino acid sequences of the human POLH and yeast Rad30 proteins are 19% identical and 31.9% similar. Masutani et al. (1999) found that POLH shares 7 conserved domains with other proteins of S. pombe, Arabidopsis thaliana, C. elegans, and S. cerevisiae. No known DNA polymerase motifs were identified in DNA polymerase eta. Northern blot analysis of RNAs from 5 XPV cell lines revealed 2 with major decreases in band intensity. Recombinant human DNA polymerase eta corrected the inability of XPV cell extracts to carry out DNA replication by bypassing thymine dimers on damaged DNA.
Johnson et al. (1999) independently cloned the human DNA polymerase eta gene, which they called hRAD30, by homology cloning of S. cerevisiae RAD30. DNA polymerase eta is involved in the error-free bypass of ultraviolet light damage.
Oxidative damage to DNA had been proposed to have a role in cancer and aging. Oxygen-free radicals formed during normal aerobic cellular metabolism attack bases in DNA, and a 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the adducts formed. Eukaryotic replicative DNA polymerases replicate DNA containing 8-oxoG by inserting an adenine opposite the lesion; consequently, 8-oxoG is highly mutagenic and causes G:C to T:A transversions. Genetic studies in yeast have indicated a role for mismatch repair in minimizing the incidence of these mutations. The gene RAD30 of S. cerevisiae encodes a DNA polymerase, Pol-eta, that efficiently replicates DNA containing a cis-syn thymine-thymine dimer by inserting 2 adenines across from the dimer. Haracska et al. (2000) showed that yeast and human POL-eta replicate DNA containing 8-oxoG efficiently and accurately by inserting a cytosine across from the lesion and by proficiently extending from this basepair. Consistent with these biochemical studies, a synergistic increase in the rate of spontaneous mutations occurs in the absence of POL-eta in the yeast ogg1 (601982) deletion mutant. Haracska et al. (2000) stated that their results suggested an additional role for POL-eta in the prevention of internal cancers in humans that would otherwise result from the mutagenic replication of 8-oxoG in DNA.
Matsuda et al. (2000) demonstrated that human POLH copies undamaged DNA with much lower fidelity than any other template-dependent DNA polymerase studied. POLH lacks an intrinsic proofreading exonuclease activity and, depending on the mismatch, makes 1 base substitution error for every 18 to 380 nucleotides synthesized. This very low fidelity indicates a relaxed requirement for correct basepairing geometry and indicates that the function of POLH may be tightly controlled to prevent potentially mutagenic DNA synthesis.
To overcome the catastrophic consequences associated with the demise of stalled replication forks, translesional synthesis polymerases promote DNA synthesis past lesions. Alternatively, a stalled fork may collapse and undergo repair by homologous recombination. McIlwraith et al. (2005) found that human POLH extended DNA synthesis past lesions following formation of a D-loop structure, with the invading strand serving as primer. POLH showed highest specificity for the D loop and weaker association with replication forks and single- and double-stranded DNA. POLH interacted with the recombinase RAD51 (179617) in HeLa cells following DNA damage, and RAD51 stimulated the DNA extension activity of POLH. Extracts from an XPV cell line exhibited severely reduced D-loop extension activity. McIlwraith et al. (2005) concluded that POLH promotes translesional synthesis at stalled replication forks and reinitiates DNA synthesis by homologous recombination repair.
B lymphocytes diversify their immunoglobulin-variable (IgV) genes through homologous recombination-mediated Ig gene conversion. Kawamoto et al. (2005) found that Polh was involved in Ig gene conversion in chicken B-lymphocyte precursors and in a chicken B-cell line. Polh -/- B cell lines exhibited hypersensitivity to ultraviolet irradiation, a significant decrease in the frequency of Ig gene conversion, and reduced double-strand break-induced homologous recombination compared with wildtype cells. Complementation with human POLH reversed these defects.
DNA polymerase eta in eukaryotes is able to replicate through UV-induced cyclobutane pyrimidine dimers (CPDs), as shown by Masutani et al. (1999) and Johnson et al. (1999, 1999). Pol-eta can also replicate through cisplatin-induced 1,2-d(GpG) cisplatin adducts (Pt-GGs) (Albertella et al., 2005) formed in a typical anticancer therapy with cisplatin (Wang and Lippard, 2005).
DNA polymerase eta performs translesion synthesis past UV photoproducts and is deficient in cancer-prone xeroderma pigmentosum variant (XPV; 278750) syndrome. The slight sensitivity of XPV cells to UV is dramatically enhanced by low concentrations of caffeine. Using DNA combing, Despras et al. (2010) showed that translesion synthesis defect led to a strong reduction in the number of active replication forks and a high proportion of stalled forks in human cells. Extensive regions of single-strand DNA were formed during replication in irradiated XPV cells, leading to an overactivation of ATR/CHK1 (601215/603078) pathway after low UVC doses. Addition of a low concentration of caffeine post-irradiation significantly decreased CHK1 activation and abrogated DNA synthesis in XPV cells. While inhibition of CHK1 activity by 7-hydroxystaurosporine (UCN-01) prevented UVC-induced S-phase delay in wildtype cells, it aggravated replication defect in XPV cells by increasing fork stalling. Consequently, UCN-01 sensitized XPV cells to UVC as caffeine did. The authors concluded that POLH acts at stalled forks to resume their progression, preventing the requirement for efficient replication checkpoint after low UVC doses. In the absence of POLH, CHK1 kinase becomes essential for replication resumption by alternative pathways, via fork stabilization.
Yuasa et al. (2000) demonstrated that the POLH gene contains 11 exons. All but 1 of the splice donor and acceptor sites contain consensus GT/AG dinucleotides; only the splice donor site in exon 11, which had the sequence CT, varied from the consensus pattern.
Crystal Structure
Alt et al. (2007) presented a structural and biochemical analysis of how Pol-eta copies Pt-GG-containing DNA. The damaged DNA is bound in an open DNA binding rim. Nucleotidyl transfer requires the DNA to rotate into an active conformation, driven by hydrogen bonding of the templating base to the dNTP. For the 3-prime dG of the Pt-GG, this step is accomplished by a Watson-Crick basepair to dCTP and is biochemically efficient and accurate. In contrast, bypass of the 5-prime dG of the Pt-GG is less efficient and promiscuous for dCTP and dATP as a result of the presence of the rigid Pt crosslink. Alt et al. (2007) concluded that their analysis revealed the structural features that enable Pol-eta to replicate across strongly distorting DNA lesions.
Silverstein et al. (2010) presented the crystal structures of S. cerevisiae POLH, also known as RAD30, in ternary complex with a cis-syn thymine-thymine (T-T) dimer and with undamaged DNA. The structures revealed that the ability of PolH to replicate efficiently through the ultraviolet-induced lesion derives from a simple and yet elegant mechanism, wherein the 2 Ts of the T-T dimer are accommodated in an active site cleft that is much more open than in other polymerases. Silverstein et al. (2010) also showed by structural, biochemical, and genetic analyses that the 2 Ts are maintained in a stable configuration in the active site via interactions with gln55, arg73, and met74. Silverstein et al. (2010) concluded that together, these features define the basis of PolH's action on ultraviolet-damaged DNA that is crucial in suppressing the mutagenic and carcinogenic consequences of sun exposure, thereby reducing the incidence of skin cancers in humans.
Biertumpfel et al. (2010) reported high-resolution crystal structures of human PolH at 4 consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. PolH acts like a molecular splint to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for 2-metal ion catalysis. Two residues conserved among PolH orthologs form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, 8 PolH missense mutations causing xeroderma pigmentosum type V (278750) can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provided an insight into the role of PolH in replicating through D loop and DNA fragile sites.
Nakamura et al. (2012) cocrystallized native human DNA polymerase eta, DNA, and dATP at pH 6.0 without Mg(2+). The polymerization reaction was initiated by exposing crystals to 1 mM Mg(2+) at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and 2 Mg(2+) ions were aligned within 40 seconds, but the bond formation was not evident until 80 seconds. From 80 to 300 seconds structures showed a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. Transient electron densities indicated that deprotonation and an accompanying C2-prime-endo to C3-prime-endo conversion of the nucleophile 3-prime-OH are rate-limiting. A third Mg(2+) ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the 2-metal-ion mechanism.
By study of a human/rodent somatic cell hybrid mapping panel followed by fluorescence in situ hybridization, Yuasa et al. (2000) mapped the POLH gene to chromosome 6p21.1-p12.
Using sequencing of cDNA from XPV cell lines, Masutani et al. (1999) identified mutations in the POLH gene in all cell lines examined; see 603968.0001 through 603968.0005.
In 4 cell lines from XPV patients, Yuasa et al. (2000) identified mutations in the POLH gene; 3 were homozygous and 1 was a compound heterozygote.
Johnson et al. (1999) identified 8 mutations in cell lines of XPV patients, 7 of which would result in severely truncated POLH protein.
Limoli et al. (2000) reported experimental results suggesting that the symptoms of elevated solar carcinogenesis in XPV patients may be associated with increased genomic rearrangements that result from double-strand breakage and rejoining in cells of the skin in which p53 (TP53; 191170) is inactivated by UV-induced mutations.
Broughton et al. (2002) analyzed mutations in the POLH gene in 21 patients with XPV who varied in age from 7 to 69 years. They identified 16 mutations that fell into 3 categories. Many of the mutations resulted in severe truncations of the protein and were effectively null alleles. However, 5 missense mutations were located in the conserved catalytic domain of the protein. Extracts of cells falling into these 2 categories were defective in the ability to carry out translesion synthesis (TLS) past sites of DNA damage. Three mutations caused truncations of the C terminus such that the catalytic domains were intact, and extracts from these cells were able to carry out TLS. Based on their previous work, however, Broughton et al. (2002) anticipated that protein in these cells would not be localized in the nucleus or would not be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations was markedly skewed toward the N-terminal half of the protein. Two of the missense mutations were predicted to affect the interaction with DNA, while the others were likely to disrupt the 3-dimensional structure of the protein. There was a wide variability in clinical features among patients, which was not obviously related to the site or type of mutation.
In the XPV (278750) cell line XP30RO, Masutani et al. (1999) identified deletion of 13 nucleotides between positions 343 and 355.
Johnson et al. (1999) reported the same 13-bp deletion as occurring at nucleotides 104-116 in cell line XP30R0 from a patient of Lebanese origin. They noted that the mutation would result in the truncation of the POLH protein at position 35. Only the mutant allele was observed in this cell line.
In the XPV (278750) cell line XP4BE, Masutani et al. (1999) identified deletion of 4 nucleotides between positions 289 and 292.
In the XPV (278750) cell line XP7TA, Masutani et al. (1999) identified deletion of 2 nucleotides at position 770-771.
In the XPV (278750) cell line XP2SA, Masutani et al. (1999) identified a G-to-T mutation at nucleotide 1153 which resulted in a glu306-to-ter mutation.
In the XPV (278750) cell line XP1RO, Masutani et al. (1999) identified a large genomic deletion that was not fully characterized. On the other allele, there was G-to-A mutation at nucleotide 1127 which resulted in a trp297-to-ter mutation.
In the XPV (278750) cell lines XP1CH and XP2CH, derived from 2 sibs of Russian-Armenian origin with XPV who were the offspring of a first-cousin marriage, Johnson et al. (1999) identified a C-to-T transition at nucleotide 376 of the POLH gene, resulting in the substitution of a premature termination codon for valine at codon 125.
In the XPV (278750) cell line XP115LO, derived from an individual of Iranian origin who was the offspring of a first-cousin marriage, Johnson et al. (1999) identified a C-to-T transition at nucleotide 1117, which created a premature termination codon in place of valine at codon 372.
In the XPV (278750) cell line XP5MA, derived from a patient of German ancestry, Johnson et al. (1999) identified a 104-bp deletion at nucleotides 661-764 of the POLH gene, resulting in a frameshift at lys220. Only the mutant allele was observed in this cell line.
In the XPV (278750) cell line XP6DU, derived from a patient of Scottish ancestry, Johnson et al. (1999) identified a mutation on each allele. One was a -1 deletion of a G at nucleotide 207, resulting in a frameshift at lys69; the other was a 3-bp deletion at nucleotides 222-224 (603968.0011), resulting in an in-frame deletion of leu at codon 75.
In the XPV (278750) cell line XP6DU, Johnson et al. (1999) identified a mutation on each allele. One mutation was a 3-bp deletion at nucleotides 222-224, resulting in an in-frame deletion of leu at codon 75; the other mutation was a frameshift at lys69 (603968.0010).
In a Japanese patient with XPV (278750) and 1 of his parents, Itoh et al. (2000) identified an A-G transition at nucleotide 1840 of the POLH cDNA, which resulted in a lys535-to-glu (K535E) missense mutation. This basic lysine residue is conserved between human and mouse (Yamada et al., 2000). The lys535-to-glu mutation likely affects the conformation and functionality of the DNA polymerase.
In a Japanese patient with XPV (278750) and 1 parent, Itoh et al. (2000) identified an A-C transversion at nucleotide 2003 of the POLH cDNA, which resulted in a lys589-to-thr (K589T) missense mutation. This basic lysine residue is conserved between human and mouse (Yamada et al., 2000). The lys589-to-thr mutation likely affects the conformation and functionality of the DNA polymerase.
Albertella, M. R., Green, C. M., Lehmann, A. R., O'Connor, M. J. A role for polymerase eta in the cellular tolerance to cisplatin-induced damage. Cancer Res. 65: 9799-9806, 2005. [PubMed: 16267001] [Full Text: https://doi.org/10.1158/0008-5472.CAN-05-1095]
Alt, A., Lammens, K., Chiocchini, C., Lammens, A., Pieck, J. C., Kuch, D., Hopfner, K.-P., Carell, T. Bypass of DNA lesions generated during anticancer treatment with cisplatin by DNA polymerase eta. Science 318: 967-970, 2007. [PubMed: 17991862] [Full Text: https://doi.org/10.1126/science.1148242]
Biertumpfel, C., Zhao, Y., Kondo, Y., Ramon-Maiques, S., Gregory, M., Lee, J. Y., Masutani, C., Lehmann, A. R., Hanaoka, F., Yang, W. Structure and mechanism of human DNA polymerase eta. Nature 465: 1044-1048, 2010. Note: Erratum: Nature 476: 360 only, 2011. [PubMed: 20577208] [Full Text: https://doi.org/10.1038/nature09196]
Broughton, B. C., Cordonnier, A., Kleijer, W. J., Jaspers, N. G. J., Fawcett, H., Raams, A., Garritsen, V. H., Stary, A., Avril, M.-F., Boudsocq, F., Masutani, C., Hanaoka, F., Fuchs, R. P., Sarasin, A., Lehmann, A. R. Molecular analysis of mutations in DNA polymerase eta in xeroderma pigmentosum-variant patients. Proc. Nat. Acad. Sci. 99: 815-820, 2002. [PubMed: 11773631] [Full Text: https://doi.org/10.1073/pnas.022473899]
Despras, E., Daboussi, F., Hyrien, O., Marheineke, K., Kannouche, P. L. ATR/Chk1 pathway is essential for resumption of DNA synthesis and cell survival in UV-irradiated XP variant cells. Hum. Molec. Genet. 19: 1690-1701, 2010. [PubMed: 20123862] [Full Text: https://doi.org/10.1093/hmg/ddq046]
Haracska, L., Yu, S.-L., Johnson, R. E., Prakash, L., Prakash, S. Efficient and accurate replication in the presence of 7,8-dihydro-8-oxoguanine by DNA polymerase eta. Nature Genet. 25: 458-461, 2000. [PubMed: 10932195] [Full Text: https://doi.org/10.1038/78169]
Itoh, T., Linn, S., Kamide, R., Tokushige, H., Katori, N., Hosaka, Y., Yamaizumi, M. Xeroderma pigmentosum variant heterozygotes show reduced levels of recovery of replicative DNA synthesis in the presence of caffeine after ultraviolet irradiation. J. Invest. Derm. 115: 981-985, 2000. [PubMed: 11121129] [Full Text: https://doi.org/10.1046/j.1523-1747.2000.00154.x]
Johnson, R. E., Kondratick, C. M., Prakash, S., Prakash, L. hRAD30 mutations in the variant form of xeroderma pigmentosum. Science 285: 263-265, 1999. [PubMed: 10398605] [Full Text: https://doi.org/10.1126/science.285.5425.263]
Johnson, R. E., Prakash, S., Prakash, L. Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase, Pol-eta. Science 283: 1001-1004, 1999. [PubMed: 9974380] [Full Text: https://doi.org/10.1126/science.283.5404.1001]
Kawamoto, T., Araki, K., Sonoda, E., Yamashita, Y. M., Harada, K., Kikuchi, K., Masutani, C., Hanaoka, F., Nozaki, K., Hashimoto, N., Takeda, S. Dual roles for DNA polymerase eta in homologous DNA recombination and translesion DNA synthesis. Molec. Cell 20: 793-799, 2005. [PubMed: 16337602] [Full Text: https://doi.org/10.1016/j.molcel.2005.10.016]
Limoli, C. L., Giedzinski, E., Morgan, W. F., Cleaver, J. E. Polymerase eta deficiency in the xeroderma pigmentosum variant uncovers an overlap between the S phase checkpoint and double-strand break repair. Proc. Nat. Acad. Sci. 97: 7939-7946, 2000. [PubMed: 10859352] [Full Text: https://doi.org/10.1073/pnas.130182897]
Masutani, C., Araki, M., Yamada, A., Kusumoto, R., Nogimori, T., Maekawa, T., Iwai, S., Hanaoka, F. Xeroderma pigmentosum variant (XP-V) correcting protein from HeLa cells has a thymine dimer bypass DNA polymerase activity. EMBO J. 18: 3491-3501, 1999. [PubMed: 10369688] [Full Text: https://doi.org/10.1093/emboj/18.12.3491]
Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M., Araki, M., Iwai, S., Takio, K., Hanaoka, F. The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase eta. Nature 399: 700-704, 1999. [PubMed: 10385124] [Full Text: https://doi.org/10.1038/21447]
Matsuda, T., Bebenek, K., Masutani, C., Hanaoka, F., Kunkel, T. A. Low fidelity DNA synthesis by human DNA polymerase-eta. Nature 404: 1011-1013, 2000. [PubMed: 10801132] [Full Text: https://doi.org/10.1038/35010014]
McIlwraith, M. J., Vaisman, A., Liu, Y., Fanning, E., Woodgate, R., West, S. C. Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination. Molec. Cell 20: 783-792, 2005. Note: Erratum: Molec. Cell 21: 445 only, 2006. [PubMed: 16337601] [Full Text: https://doi.org/10.1016/j.molcel.2005.10.001]
Nakamura, T., Zhao, Y., Yamagata, Y., Hua, Y., Yang, W. Watching DNA polymerase eta make a phosphodiester bond. Nature 487: 196-201, 2012. [PubMed: 22785315] [Full Text: https://doi.org/10.1038/nature11181]
Silverstein, T. D., Johnson, R. E., Jain, R., Prakash, L., Prakash, S., Aggarwal, A. K. Structural basis for the suppression of skin cancers by DNA polymerase eta. Nature 465: 1039-1043, 2010. [PubMed: 20577207] [Full Text: https://doi.org/10.1038/nature09104]
Wang, D., Lippard, S. J. Cellular processing of platinum anticancer drugs. Nature Rev. Drug Discov. 4: 307-320, 2005. [PubMed: 15789122] [Full Text: https://doi.org/10.1038/nrd1691]
Yamada, A., Masutani, C., Iwai, S., Hanaoka, F. Complementation of defective translesion synthesis and UV light sensitivity in xeroderma pigmentosum variant cells by human and mouse DNA polymerase eta. Nucleic Acids Res. 28: 2473-2480, 2000. [PubMed: 10871396] [Full Text: https://doi.org/10.1093/nar/28.13.2473]
Yuasa, M., Masutani, C., Eki, T., Hanaoka, F. Genomic structure, chromosomal localization and identification of mutations in the xeroderma pigmentosum variant (XPV) gene. Oncogene 19: 4721-4728, 2000. [PubMed: 11032022] [Full Text: https://doi.org/10.1038/sj.onc.1203842]