Schizosaccharomyces pombe AGT-homolog's (SpAho) role in DNA repair of alkylated lesions



Matthew Evans, Class of 2007
Box 649

Primary Sponsor

Anthony E. Pegg, Ph.D.
Evan Pugh Professor of Cellular and Molecular Physiology, and Pharmacology
Department of Cellular and Molecular Physiology
Milton S. Hershey Medical Center/Penn State College of Medicine

Research Period

May 25-August 13, 2004


To characterize the mechanism of action of the S. pombe version of the AGT-like protein and its effect/role in binding to O6-alkylguanine lesions in DNA and their repair.


O6-alkylguanine DNA-alkyltransferase (AGT) is an important protein involved in the DNA repair of alkylation damage at the O6  position of guanine and O4 position of thymine caused by endogenous and exogenous compounds (Sedgwick et al., 2002). Repair of such DNA alkylation by AGT is vital since O6 -alkylguanine and O4-alkylthymine are highly mutagenic, often resulting in GCàAT and ATà GC transition mutations during DNA replication ( Pauly et al., 1995). All AGT proteins have an active site with the sequence of -ProCysHisArgVal(Ile)- in which the alkyl group from O6-alkylguanine and O4-alkylthymine in damaged DNA is transferred, in an irreversible and stoichiometric reaction, to the active site cysteine (Pegg et al., 1995).

The function of AGT has been studied most extensively in Escherichia coli, where two AGT proteins, a 39kDa inducible protein (Ada) and a 19kDa constitutively expressed protein (Ogt), have been identified (Sedgwick, 2004). In particular, the Ada protein has two major domains involved in DNA repair that function independently. The specific domain of interest is the C-terminal domain that is involved in the transfer of alkyl groups from damaged bases, such as O6-alkylguanine, to the active site Cys-321 by a rapid but suicidal reaction (Demple et al., 1985). This reaction is currently believed to occur through the binding of damaged DNA through the enzyme's ‘helix-turn-helix wing' motif and then alignment of the damaged base with the active site cysteine through a nucleotide flipping mechanism (Daniels et al., 2004; Daniels et al., 2000).

Human AGT plays a major role in alkylation chemotherapy of tumor cells, in which tumor cells are bombarded with alkylating agents that cause excessive alkylation of DNA eventually leading to cell killing through apoptosis. However, human tumors can become resistant to this alkylation therapy due to over-expression of AGT (Mattern et al., 1998). In an attempt to inhibit this over-expression, O6 –benzylguanine, an inhibitor that was first shown to inactivate human AGT in vitro, can be used in the clinic (Pegg et al., 1993). However, the use of O6 –benzylguanine can also lead to suppression of AGT in hematopoietic stem cells, making them increasingly susceptible to alkylation (Koc ON et al., 1996). The use of gene therapy to overcome this myelosuppression is being examined elsewhere.

AGT has been found to have homologs in a variety of other organisms as well. However, S. pombe, whose entire genome is fully sequenced, does not contain such a protein, but still has the ability to survive alkylation damage to its DNA (Memisoglu et al., 2000). Genome sequencing and species comparison has discovered an AGT-like protein in S. pombe, and many other organisms including E.coli, that contains a tryptophan in place of the cysteine in its active site, referred to as Aho (AGT homolog). Otherwise, the Aho protein is relatively homologous to normal AGT protein found in other species, including a similar DNA binding domain. This project seeks to characterize this S. pombe AGT-like protein and how it might function in repairing alkylation DNA damage. The current hypothesis is that this protein has the ability to recognize and bind to alkylation damage on DNA, but ultimately does not have the ability to transfer the alkylation from the DNA to the protein itself as seen with normal AGT. One hypothesis is that the protein recognizes alkylation damage and recruits the nucleotide excision repair pathway to this complex, resulting in DNA damage repair.

The overall goal of the project is to ascertain a better understanding of alkylation DNA damage repair with hopes that it can be used to better treat patients in the future. Although a direct clinical use for the knowledge of how the S. pombe AGT-like protein (SpAho) is not readily available, gaining a better understanding of how damaged DNA is repaired in this organism will ultimately lead to better understanding of the very important role DNA damage repair has in all living organisms. There is also the possibility that humans, although not yet identified, could have a similar AGT-like protein and understanding how the S. pombe version works could prove extremely beneficial.


A DNA clone (SPAC1250.04) that codes for SpAho was obtained from The Wellcome Trust Sanger-Institute, UK. The S. pombe AGT homolog (SpAho) will be amplified from this clone via PCR using specially designed primers. The cloned product will then be purified and treated with EcoRI and Bam HI restriction enzymes allowing for ligation into the expression vector, pQE30. The expression vector will also provide a 6 histidine tag at the C-terminal end of the SpAho protein to allow for subsequent protein purification.

The ligated vector will be transformed into XL-1 Blue competent E. coli cells and grown on LB-Amp agar plates. Random colonies will be selected and cultured in LB-Amp medium and the plasmid DNA will be isolated from a specific volume of these cultures. A restriction reaction will be performed on the isolated plasmid using EcoRI and Bam HI restriction enzymes to verify that the plasmid has the SpAho insert (pQE-SpAho). Sequencing of the isolated pQE-SpAho will also be performed to verify that the nucleotide sequence of SpAho is correct and no secondary mutations were introduced during plasmid construction.

Three liters of E. coli cells containing pQE-SpAho will be grown and the SpAho protein will be induced with IPTG. The E.coli cells will be lysed by sonication and the cleared lysate containing SpAho protein (if soluble) will be purified by passing through a Talon resin column that binds histidine tagged proteins. The purity of the isolated protein will be checked on a SDS PAGE gel and the purified protein will be tested for alkytransferase activity by examining either the disappearance of radioactive alkyl groups from O6 position of guanine in DNA or the transfer of radioactive alkyl groups from DNA to the purified protein.

In addition, the SpAho protein will be expressed in an E. coli strain (GWR109) that lacks its two AGT proteins (Ada and Ogt) to test if this protein provides protection to cells from alkylating agents. The protein will also be expressed in two different strains of E.coli, one with and one without a nucleotide excision repair pathway, that also lack both AGT proteins. If NER is involved, a significant difference should be seen between the two cell lines in terms of protection from alkylating agent MNNG.

Student Responsibilities

  1. Review literature on O6-alkylguanine DNA-alkyltransferase, its function and role in chemotherapy
  2. Carry out experiments as outlined in Methods Section
  3. Prepare draft and final MSR paper


Sponsor Responsibilities

  1. Provide guidance in project development and implementation
  2. Provide access to equipment and reagents and necessary training in the operation of equipment and procedures
  3. Review draft and final MSR paper



Daniels DS, Mol CD, Arvai A, Kanugula S, Pegg AE and Tainer JA. (2000) Active and alkylated human AGT structures: a novel zinc site, inhibitor and extrahelical base binding. The EMBO Journal. 19(7):1719-1730.

Daniels DS, Woo TT, Luu KX, Noll DM, Clarke ND, Pegg AE and Tainer JA (2004) DNA binding and nucleotide flipping by the human DNA repair protein AGT. Nature Structural and Molecular Biology. 11(8): in press

Demple B, Sedgwick B, Robins P, Totty N, Waterfield MD and Lindahl T. (1985) Active site and complete sequence of the suicidal methyltransferase that counters alkylation mutagenesis. Proc. Natl. Acad. Sci. USA. 82:2688-2692.

Koc ON, Phillips WP Jr, Lee K, Liu L, Zaidi NH, Allay JA, Gerson SL. (1996) Role of DNA repair in resistance to drugs that alkylate O6 of guanine. Cancer Treat. Res. 87:123-46.

Mattern J, Eichorn U, Kaina B and Volm M. (1998) O6 –methyl-guanine-DNA methyltransferase activity and sensitivity to cyclophosphamide and cisplatin in human lung tumor xenografts. Int. J. Cancer. 77:919-922.

Memisoglu A, Samson L. (2000) Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe. J Bacteriol. 182(8): 2104-2112.

Pauly GT, Hughes SH., Moschel RC. 1995 Mutagenesis in Escherichia coli by three O6 –substituted guanines in double-stranded or gaped plasmids. Biochemistry. 34:8924-8930.

Pegg AE, Boosalis M, Samson L, Moschel RC, Byers TL, Swenn K and Dolan ME. (1993) Mechanism of inactivation of human O6 –alkylguanine-DNA alkyltransferase by O6 –Benzylguanine. Biochemistry. 32: 11998-12006.

Pegg AE, Dolan ME, Moschel RC. (1995) Structure, function and inhibition of O6 –alkylguanine DNA alkyltransferase. Prog. Nucleic Acid Res. Mol. Biol. 51:167-223.

Sedgwick B, Lindahl, T. (2002) Recent progress on the Ada response for inducible repair of DNA alkylation damage. Oncogene. 21:8886-8894.

Sedgwick B. (2004) Repairing DNA-methylation damage. Natl Rev Mol Cell Biol. 2:148-157


Anthony E. Pegg, Ph.D. Date

Matthew S. Evans Date