Fidelity and Damage Bypass Ability of Schizosaccharomyces pombe Eso1 Protein, Comprised of DNA Polymerase η and Sister Chromatid Cohesion Protein Ctf7

DNA polymerase η (Polη) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Polη in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). BothSaccharomyces cerevisiae and human Polη efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeastSchizosaccharomyces pombe, theeso1 + encoded protein is comprised of two domains, wherein the NH2 terminus is highly homologous to Polη, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Polη domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of ∼10−2 to 10−3. We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Polη. The possible role of Ctf7 fusion with Polη in the replication of Cohesin-bound DNA sequences is discussed. DNA polymerase η (Polη) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Polη in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). BothSaccharomyces cerevisiae and human Polη efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeastSchizosaccharomyces pombe, theeso1 + encoded protein is comprised of two domains, wherein the NH2 terminus is highly homologous to Polη, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Polη domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of ∼10−2 to 10−3. We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Polη. The possible role of Ctf7 fusion with Polη in the replication of Cohesin-bound DNA sequences is discussed. glutathioneS-transferase cyclobutane pyrimidine dimer In Saccharomyces cerevisiae as well as humans, DNA polymerase η functions in error-free replication of UV-damaged DNA. In S. cerevisiae, inactivation of Polη confers enhanced UV sensitivity and leads to an increase in UV-induced mutation frequencies (1McDonald J.P. Levine A.S. Woodgate R. Genetics. 1997; 147: 1557-1568Crossref PubMed Google Scholar, 2Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1999; 274: 15975-15977Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 3Yu S.-L. Johnson R.E. Prakash S. Prakash L. Mol. Cell. Biol. 2001; 21: 185-188Crossref PubMed Scopus (120) Google Scholar). In humans, inactivation of Polη results in the cancer prone syndrome, the variant form of xeroderma pigmentosum (4Johnson R.E. Kondratick C.M. Prakash S. Prakash L. Science. 1999; 285: 263-265Crossref PubMed Scopus (673) Google Scholar, 5Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1153) Google Scholar). Cells from variant form of xeroderma pigmentosum patients display a deficiency in the replication of UV-damaged DNA (6Lehmann A.R. Kirk-Bell S. Arlett C.F. Paterson M.C. Lohman P.H.M. de Weerd-Kastelein E.A. Bootsma D. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 219-223Crossref PubMed Scopus (525) Google Scholar, 7Cleaver J.E. Greene A.E. Coriell L.L. Mulivor R.A. Cytogenet. Cell Genet. 1981; 31: 188-192Crossref PubMed Scopus (22) Google Scholar, 8Boyer J.C. Kaufmann W.K. Brylawski B.P. Cordeiro-Stone M. Cancer Res. 1990; 50: 2593-2598PubMed Google Scholar), and they are hypermutable with UV light (9Wang Y.-C. Maher V.M. Mitchell D.L. McCormick J.J. Mol. Cell. Biol. 1993; 13: 4276-4283Crossref PubMed Scopus (142) Google Scholar, 10Waters H.L. Seetharam S. Seidman M.M. Kraemer K.H. J. Invest. Dermatol. 1993; 101: 744-748Abstract Full Text PDF PubMed Google Scholar). Polη is unique among eukaryotic DNA polymerases in its proficient ability to replicate through DNA lesions which distort the DNA helix (5Masutani C. Kusumoto R. Yamada A. Dohmae N. Yokoi M. Yuasa M. Araki M. Iwai S. Takio K. Hanaoka F. Nature. 1999; 399: 700-704Crossref PubMed Scopus (1153) Google Scholar, 11Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (696) Google Scholar, 12Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 13Haracska L., Yu, S.-L. Johnson R.E. Prakash L. Prakash S. Nat. Genet. 2000; 25: 458-461Crossref PubMed Scopus (306) Google Scholar, 14Haracska L. Prakash S. Prakash L. Mol. Cell. Biol. 2000; 20: 8001-8007Crossref PubMed Scopus (118) Google Scholar). Both yeast and human Polη replicate through acis-syn thymine-thymine (T-T) dimer with the same efficiency and accuracy as they replicate through the two undamaged Ts (12Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 15Washington M.T. Johnson R.E. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3094-3099PubMed Google Scholar). The ability of Polη to insert nucleotides opposite distorting DNA lesions and to carry out extension of the nascent DNA strand has suggested that Polη is more tolerant of geometric distortions in DNA than are other DNA polymerases which cannot bypass DNA lesions. Accordingly, both S. cerevisiae and human Polη are low fidelity enzymes, misincorporating nucleotides with a frequency of 10−2 to 10−3 (12Johnson R.E. Washington M.T. Prakash S. Prakash L. J. Biol. Chem. 2000; 275: 7447-7450Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 16Washington M.T. Johnson R.E. Prakash S. Prakash L. J. Biol. Chem. 1999; 274: 36835-36838Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 17Matsuda T. Bebenek K. Masutani C. Hanaoka F. Kunkel T.A. Nature. 2000; 404: 1011-1013Crossref PubMed Scopus (327) Google Scholar). Although Polη is a low fidelity enzyme, it does not contribute to spontaneous mutagenesis, since the rate of spontaneous mutations at several loci examined remains the same in the presence or absence of Polη in S. cerevisiae(18Pavlov Y.I. Nguyen D. Kunkel T.A. Mutat. Res. 2001; 478: 129-139Crossref PubMed Scopus (44) Google Scholar). 1R. E. Johnson, L. Prakash, and S. Prakash, unpublished observations. Thus, Polη may have little or no effect on normal replication, and its function may be primarily restricted to promoting replication through DNA lesions. The Rad6-Rad18 complex, comprised of the ubiquitin conjugating and DNA binding activities (19Bailly V. Lamb J. Sung P. Prakash S. Prakash L. Genes Dev. 1994; 8: 811-820Crossref PubMed Scopus (280) Google Scholar, 20Bailly V. Lauder S. Prakash S. Prakash L. J. Biol. Chem. 1997; 272: 23360-23365Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), may be a key factor in limiting Polη action to damage bypass. Although the mechanism of the Rad6-Rad18 enzyme complex remains unknown, it is possible that ubiquitin conjugation by the Rad6-Rad18 complex leads to dissociation of some protein(s) from the replication machinery stalled at a lesion site, and that, in turn, promotes the assembly of a trans-lesion DNA synthesis polymerase such as Polη into the stalled replication complex. Alternatively, Polη could be kept in an inactive state by its binding to other protein(s), and upon the infliction of damage to DNA, the Rad6-Rad18 mediated protein ubiquitination may stimulate the dissolution of the inhibitory protein(s), thereby activating Polη. Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1+ -encoded protein is comprised of two domains, of which the NH2-terminal two-thirds is highly homologous to S. cerevisiae and human Polη, and the COOH-terminal one-third is highly homologous to theS. cerevisiae Ctf7 protein (also called Eco1) (21Tanaka K. Yonekawa T. Kawasaki Y. Kai M. Furuya K. Iwasaki M. Murakami H. Yanagida M. Okayama H. Mol. Cell. Biol. 2000; 20: 3459-3469Crossref PubMed Scopus (160) Google Scholar), an essential protein required for the establishment of sister chromatid cohesion during S phase (21Tanaka K. Yonekawa T. Kawasaki Y. Kai M. Furuya K. Iwasaki M. Murakami H. Yanagida M. Okayama H. Mol. Cell. Biol. 2000; 20: 3459-3469Crossref PubMed Scopus (160) Google Scholar, 22Skibbens R.V. Corson L.B. Koshland D. Hieter P. Genes Dev. 1999; 13: 307-319Crossref PubMed Scopus (385) Google Scholar, 23Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (502) Google Scholar). Deletion analyses have indicated that the COOH-terminal Ctf7 portion of Eso1 is sufficient and necessary for sister chromatid cohesion, whereas deletion of the NH2-terminal Polη portion increases the sensitivity to UV irradiation but has no effect on sister chromatid cohesion (21Tanaka K. Yonekawa T. Kawasaki Y. Kai M. Furuya K. Iwasaki M. Murakami H. Yanagida M. Okayama H. Mol. Cell. Biol. 2000; 20: 3459-3469Crossref PubMed Scopus (160) Google Scholar). Thus, although the two proteins are encoded by the same gene, they retain their respective functions in sister chromatid cohesion and damage bypass. The fusion of Polη with Ctf7 in S. pombe raised the possibility that although the two proteins are encoded by separate genes in other species, even there these proteins may associatein vivo, and that association may modulate the function of one or both the proteins. For instance, it could be that association with Ctf7 inactivates Polη and its activation requires that the two proteins dissociate following the Rad6-Rad18-dependent ubiquitination of one or both proteins; alternatively, association with Ctf7 could improve the fidelity and processivity of Polη. Here, we purify the S. pombe eso1+ -encoded protein containing both the Polη and Ctf7 domains, and compare the DNA synthesis properties of Polη alone and of Polη fused to Ctf7 in Eso1. Unexpectedly, we find that fusion with Ctf7 has no effect on Polη's DNA synthesis or damage bypass ability, or on its fidelity or processivity. We discuss these observations in relation to the possible role of Polη's association with Ctf7. To obtain the full-length Eso1-(1–872) protein and the truncated Eso1-(1–609) protein which lacks the Ctf7 domain but contains the entire Polη domain, herein referred to as Polη, theeso1+ gene was amplified from the S. pombe strain JFP41 (h+ ura4-D18 ade6-M216 leu1–32) total genomic DNA by polymerase chain reaction using oligonucleotide N7119 (5′-CAGGGGTACCGGATCCACATATGGAATTAGGCAAAAGCAAATTCTC-3′) and the oligonucleotide N7118 (5′-GGTCGTCGACGGATCCTCAACTTTCATAAACAGCATATCGAAG-3′) or the oligonucleotide N7117 (5′-CAGGGTCGACGGATCCTCATCTTTTGTTGTTTGTTTCATCGG-3′), respectively. The amplified DNAs were then digested with Asp-718 and SalI restriction endonucleases and cloned into YIplac211, generating plasmids pBJ796 and pBJ800, respectively. The cloned polymerase chain reaction fragments in pBJ796 and pBJ800 were sequenced and found not to contain any mutations. Subsequently, the wild type and truncatedeso1 genes were cloned in-frame with the glutathioneS-transferase (GST)2 gene under the control of a galactose-inducible phosphoglycerate kinase promoter in plasmid pBJ760 (24Johnson R.E. Washington M.T. Haracska L. Prakash S. Prakash L. Nature. 2000; 406: 1015-1019Crossref PubMed Scopus (578) Google Scholar), generating the expression vectors pBJ808 and pBJ811, respectively. The GST-tagged proteins were expressed in the S. cerevisiae strain BJ5464 harboring either the plasmid pBJ808 or pBJ811. S. cerevisiae cells were grown overnight in synthetic complete medium lacking leucine (SC-leu) and containing 2% dextrose, 2.5% lactate, and 3% glycerol before diluting 30-fold in SC-leu as above but lacking dextrose. After 16 h, 2% galactose was added and cells were grown for an additional 7 h before harvesting by centrifugation. To purify GST-Eso1 and GST-Polη proteins, cells were resuspended in 2 volumes of ice-cold cell breakage buffer (50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 300 mm NaCl, 10% sucrose, 0.5 mm benzamidine, 0.5 mm phenylmethylsulfonyl fluoride, and one Complete Protease Inhibitor Mixture Tablet per 50 ml of extract (Roche Molecular Biochemicals)), and lysed at 4 °C in a French Press at 120,000 kilopascal. Cell debris was removed by centrifugation at 100,000 × g, and cell extract was passed over a 100 μl of glutathione-Sepharose 4B column (Amersham Pharmacia Biotech) at 4 °C. The beads were washed with 10 volumes ice-cold cell breakage buffer containing 1 m NaCl, followed by 5 volumes ice-cold low salt buffer (50 mmTris-HCl, pH 7.5, 1 mm EDTA, 50 mm NaCl, 10% The GST-Eso1 and the GST-Polη proteins were at 4 °C with 100 μl of buffer mm Tris-HCl, pH 7.5, 100 mm NaCl, 10% mm of protein were at °C. For the fidelity of we the following as which only in the To we the following For replication in the presence of a dimer we the following as the The template a thymine-thymine (T-T) dimer at the whereas the control template two undamaged We the following for bypass were using kinase (Roche Molecular and (Amersham Pharmacia or were to or in the presence of 50 mm Tris-HCl, pH 7.5, and 100 mm by the to °C for 2 and to °C over several The DNA polymerase mm Tris-HCl, pH 7.5, 100 5 mm 5 and 10% and was out for 5 at °C. For DNA a 50 and 100 of of the were and were by the of to of GST-Eso1 or GST-Polη proteins. were in 10 volumes of buffer 10 mm EDTA, pH 1 1 at °C for 2 on and on 10% containing m DNA polymerase fidelity were out under but 50 or and of a to The were by the of 5 GST-Eso1 or and were 2 to in 10 volumes of of the and were using a with dimer bypass were out under with 10 template and no one of the at 100 or at 100 The were by the of 5 GST-Eso1 or and 5 in 10 volumes of For of the of the was by the reaction in the rate of was as a function of and the were by using to the 1 and m steady-state kinetic for the of the and were from the were to the frequency of as S. L.B. J. Mol. Biol. 1993; PubMed Scopus Google Scholar, S. L.B. PubMed Scopus Google using the following 2 was by 50 GST-Eso1 or GST-Polη with 50 DNA under for 1 were by the of of and 1 DNA as a To the of the we a control reaction in which GST-Eso1 or GST-Polη was with the DNA and the for 1 h before the of After or were and as above for the the processivity, is the that for the polymerase to at one additional N. Y. Acad. Sci. 1994; PubMed Scopus Google Scholar). To the processivity of and Polη, we the of the at the reaction using the we the of polymerase that at which is the of the of than or to the to to the total of the is expressed in the following is the of In a DNA binding it with a at 1 to any N. Thus, the the of polymerase that at and the processivity, is expressed in the following 4 we the of polymerase that at and a for the from the to 4 using The of nucleotides per DNA binding was then The two-thirds of the S. pombe eso1+ -encoded protein and similar with the S. cerevisiae Polη, and the COOH-terminal one-third of the protein and similar with the S. cerevisiae Ctf7 protein (21Tanaka K. Yonekawa T. Kawasaki Y. Kai M. Furuya K. Iwasaki M. Murakami H. Yanagida M. Okayama H. Mol. Cell. Biol. 2000; 20: 3459-3469Crossref PubMed Scopus (160) Google Scholar), which is highly among and an essential role in the establishment of sister chromatid cohesion during DNA replication (21Tanaka K. Yonekawa T. Kawasaki Y. Kai M. Furuya K. Iwasaki M. Murakami H. Yanagida M. Okayama H. Mol. Cell. Biol. 2000; 20: 3459-3469Crossref PubMed Scopus (160) Google Scholar, 22Skibbens R.V. Corson L.B. Koshland D. Hieter P. Genes Dev. 1999; 13: 307-319Crossref PubMed Scopus (385) Google Scholar, 23Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (502) Google Scholar). the Eso1 and the S. cerevisiae Polη and Ctf7 proteins. are in one of which to the the COOH terminus of Polη and the other to the the NH2 terminus of Ctf7. The highly found in the protein R.E. Washington M.T. Prakash S. Prakash L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), are in the Polη portion of Eso1. To for the of the COOH-terminal portion on the polymerase activity of Eso1 we expressed in S. cerevisiae both the full-length Eso1 protein and a truncated Eso1 protein that contains only the NH2-terminal and which to the Polη domain, as with the The GST-Eso1 and GST-Polη proteins were to 2 and their DNA synthesis and damage bypass properties were To for DNA polymerase activity of S. pombe Eso1 and Polη proteins, of proteins were with DNA in the presence of and reaction were on a in both proteins exhibit DNA the that a polymerase the the opposite a template We steady-state to the fidelity pombe Eso1 and Polη proteins opposite undamaged template as under and The of either the or was and to and m S. pombe Eso1 protein was with the DNA and of one of the in a in to examine the opposite template the of and was from to and from to respectively. The of by S. pombe Eso1 protein opposite the template are in were to the and to the m in the for the Eso1 protein from × to × 10−3. The were for the Polη in Polη nucleotides with the same frequencies as the Eso1 the of and opposite and by S. pombe Eso1 and Polη proteins. that the two proteins nucleotides with the same Thus, the presence of the Ctf7 protein in the COOH terminus of Eso1 has effect on Polη's ability to the or nucleotides opposite undamaged template of S. pombe Eso1 protein on undamaged × × × × × × × × × × × × × × × × × × × × × × × × 10−3 in a of S. pombe Polη on undamaged × × × × × × × × × × × × × × × × × × × × × × × × in a of efficiency of by S. pombe Eso1 and Polη proteins. The efficiency of of of the nucleotides opposite template and is for Eso1 and Polη proteins. In the the the and the is in the is a of the of a polymerase in a DNA binding is expressed as the that following the polymerase to at one additional N. Y. Acad. Sci. 1994; PubMed Scopus Google Scholar). To that we were the activity of a DNA binding we DNA to any polymerase that from the DNA. The Eso1 or Polη 5 proteins were with DNA for 1 h before the of DNA and To that the DNA was sufficient to the of Eso1 or Polη proteins to the DNA the Eso1 5 or Polη 5 proteins were with the DNA and the DNA for 1 h before the of The of any DNA synthesis in these 5 the of DNA to Eso1 or Polη The of the at the were to the and the of nucleotides per DNA binding and 5 a of the of polymerase that at for the Eso1 the to we a to which that for the Eso1 protein has a of to at one additional Thus, the of nucleotides by the Eso1 protein per DNA binding is 5 is the for Polη. the to we a for to Thus, the of nucleotides by Polη per DNA binding is The ability of Eso1 and Polη proteins to replicate through acis-syn dimer was using a in both proteins are to a opposite the of the dimer and to from and the DNA synthesis activity on the DNA is as as on the DNA. To the by Eso1 and Polη proteins opposite the two Ts of the we enzyme for 5 in the presence of dimer containing DNA and containing a or at 100 in both proteins primarily an opposite of the Ts of the dimer. The fusion of Polη to the sister chromatid cohesion protein Ctf7 in the S. pombe Eso1 protein has the to the fusion with Ctf7 Polη results that the DNA synthesis activity of Polη is not by fusion to as the Eso1 and Polη proteins DNA with similar and and on undamaged DNA, they misincorporate nucleotides with frequencies from 2 × 10−2 to 2 × the two proteins replicate a dimer equally well and both insert two opposite the two Ts of the dimer. In these the S. pombe Eso1 and Polη proteins S. cerevisiae and human Polη. Polη is a low processivity enzyme, from DNA DNA polymerases synthesis by with a in or in polymerase increases its processivity by a complex with E. P. J. Biol. Chem. 1975; Full Text PDF PubMed Google Scholar, S. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The processivity of Polη, is not by its fusion to Ctf7. Both the Eso1 and Polη proteins exhibit low processivity, nucleotides per DNA binding Although Ctf7 does not or the DNA activity or dimer bypass ability of Polη, fusion with Ctf7 may Polη to function in sister chromatid in S. cerevisiae have suggested that a cohesion complex of the and is at the of although not a is required for the association of the cohesion complex with A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (502) Google Scholar). The is a of the complex is it required for the association of with A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (502) Google Scholar). is essential for the establishment of cohesion during DNA replication, but it is not required for the of cohesion during and R.V. Corson L.B. Koshland D. Hieter P. Genes Dev. 1999; 13: 307-319Crossref PubMed Scopus (385) Google Scholar, 23Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (502) Google Scholar). possible role for the fusion of Ctf7 with Polη in the Eso1 protein is that it promotes replication through has DNA, and replication through such may be a for the of protein Cohesin-bound sister the above the of Ctf7 in S. cerevisiae and S. pombe for the establishment of sister chromatid cohesion during S it to for the that deletion of Polη has no effect on sister chromatid cohesion in either yeast possible for is the of two other highly proteins, and in sister chromatid is to complete S and results in of cohesion the sister de A. C. Science. 2000; PubMed Scopus (163) Google Scholar). The proteins are of the and a DNA polymerase activity has in de A. C. Science. 2000; PubMed Scopus (163) Google Scholar). role for the and polymerases in the replication of Cohesin-bound DNA has de A. C. Science. 2000; PubMed Scopus (163) Google Scholar). The that the and proteins are essential for the establishment of sister chromatid cohesion that these polymerases are for the replication of Cohesin-bound DNA whereas the of Polη for sister chromatid cohesion that protein a role in the replication of Cohesin-bound DNA. Polη may have an role in the replication of Cohesin-bound DNA, it promotes replication through some by the or Polη may at such in a to its role in damage bypass it the replication stalled at a lesion