- Email: [email protected]
DNA polymerase β
The International Journal of Biochemistry & Cell Biology 34 (2002) 321–324
Molecules in focus
DNA polymerase  Haitham T. Idriss a,∗ , Osama Al-Assar b , Samuel H. Wilson a a
Laboratory of Structural Biology, NIEHS, NIH, Research Triangle Park, North Carolina, NC 29907, USA b Biomedical Research Centre, Medical School, University of Dundee, Ninewells Hospital Dundee DD1 9SY, Dundee, UK Received 20 July 2001; accepted 4 October 2001
Abstract Mammalian DNA polymerase (-pol) is a single polypeptide chain enzyme of 39 kDa. -pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of -pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the -pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of -pol’s role in protecting genomic DNA. © 2002 Published by Elsevier Science Ltd. Keywords: DNA synthesis; DNA polymerase ; Base excision repair
1. Introduction Living cells face the tremendous task of maintaining an intact genome during their life span. This is necessary for cells to function in a complex environment, divide at the required time and die when appropriate. DNA synthesis is therefore necessary to duplicate the genome before cell division commences. DNA synthesis is also necessary during DNA repair. DNA replication is carried out by enzymes called DNA polymerases (DNA pols) [1]. The number of DNA pols has increased since the initial discovery of DNA pol ␣ in eukaryotic cells in 1957. In the early 1970s, DNA pol  and ␥ were discovered. This led to the immediate assumption that DNA pol ␣,  and ␥ are involved in DNA replication, repair and mitochon∗ Corresponding author. Tel.: +1-919-1-541-2644; fax: +1-919-1-541-4724. E-mail address: [email protected] (H.T. Idriss).
drial DNA replication, respectively. The discovery of DNA pol ␦ and ε during the 1980s complicated this interpretation. It was later suggested that DNA pols may serve more than one function [2]. DNA pols often have complex polypeptide structures (Fig. 1). In addition to the DNA polymerising function, which is often associated with a proofreading 3 → 5 exonuclease activity, DNA pols assume other important roles, such as DNA primase activity. Other functions include the ability to facilitate DNA polymerase interaction with other proteins involved in checkpoint function, cell cycle control, and DNA replication or repair [3]. DNA pols appear to possess a common catalytic active site. A two-metal ion catalysed mechanism guarantees the incorporation of the appropriate deoxyribonuleoside triphosphate base. However, DNA pols differ in various aspects of their structural architecture and it can be concluded that the active site of the DNA pol is conserved, whereas, the structure of
1357-2725/02/$ – see front matter © 2002 Published by Elsevier Science Ltd. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 1 3 1 - 5
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Fig. 1. Genomic structure of DNA polymerase , illustrating the major domains in the protein molecule.
Fig. 2. Crystallographic structure for DNA polymerase  complexed with gapped DNA substrate. Image prepared using GRASP [23].
H.T. Idriss et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 321–324
the surface of the molecules might differ considerably [4,5]. 2. DNA polymerase  structure Pol  is a 39 kDa single chain polypeptide comprising 335 amino acid residues (Fig. 1). Experimental studies have shown that the enzyme is folded into distinct domains each associated with a specific functional activity. The amino-terminus (8 kDa) is connected to the polymerase domain (31 kDa) by a protease sensitive hinge region [6] Fig. 2. The 8 kDa domain has a lyase activity that removes the 5-deoxyribose phosphate generated after incision by an apurinic/apyrimidinic (AP) endonulcease during base excision repair (BER) [7,8]. The nucleotidyl transferase reaction is catalysed by the 31 kDa polymerase domain. In addition to the significant movement of the amino-terminus domain upon binding gapped DNA, the C-terminus is observed to ‘close’ around the correct incoming dNTP and its complementary template base [9,10]. 3. Roles of DNA pol  DNA pol  has been shown to be primarily involved in DNA repair. There are two biochemical pathways of BER in mammalian cells: short-patch (single nucleotide replacement) and long-patch BER (multi-nucleotide replacement) [4]. Experimental evidence indicates that DNA pol  is involved in short-patch BER. In addition, DNA pol ␦ or ε are probably involved in PCNA-dependent, long-patch BER with gaps of 2–13 nucleotides [11,12]. In this pathway, the flap endonuclease 1 (Fen1) is needed to cleave a reaction intermediate generated by template strand displacement during gap filling. A variation of the long-patch BER pathway with gap lengths of 2–6 nucleotides has also been characterised in mammalian cells. This Fen1 and PCNA-dependent pathway can be reconstituted with DNA pol ␦ and DNA pol ε. However, lack of DNA pol  in DNA pol  deficient cells or in the presence of neutralising antibody, causes a reduction in DNA repair activity. This strongly suggests a role for DNA pol  in this pathway in vivo [13].
323
Another role for DNA pol  is in meiosis and double strand break repair. DNA pol  has been implicated in the meiotic events associated with synapsis and recombination [14]. In addition, a 67 kDa homologue of mammalian DNA pol , encoded by the non-essential POL4 gene of S. cerevisiae, has recently been implicated in double strand break repair through a non-homologous end joining mechanism [15]. 4. DNA Pol  and BER Recent evidence has confirmed a role for DNA pol  in mammalian AP site BER. The working model for the short-patch BER pathway is as follows [16,17]. The glycosidic bond linking the damaged base and deoxyribose is cleaved either spontaneously or by a DNA glycosylase activity removing the inappropriate base to generate an abasic or AP site in double stranded DNA. The backbone of the phosphodiester of the AP site is cleaved 5 to the sugar moiety by AP endonuclease. This leaves a 3 -hydorxyl group and a deoxyribose phosphate group at the 5 -terminus. Subsequently, excision of the deoxyribose phosphate group is catalysed by 2-deoxyribose-5-phosphate lyase, an activity which is intrinsic to the amino-terminus of the DNA pol . The gap is filled by DNA pol  and DNA ligase seals the resulting nick. In addition to its dRlyase catalytic function, the 8 kDa N-terminus carries single strand DNA binding activity. The DNA binding specificity directs the enzyme to short gaps possessing a 5 -phosphate at the margin of the gap [18]. If the 5 -phosphate containing gap is one to six nucleotides, the enzyme binds and performs DNA synthesis in a processive fashion to completely fill the gap. However, if the gap size is larger than six nucleotides, the enzyme binds at the 5 margin of the gap but performs no DNA synthesis [19]. Therefore, processive DNA synthesis is coordinated by the 8 kDa domain of DNA pol  for a gap of six nucleotides or less. Several studies have shown DNA pol  to be closely associated with DNA ligase [18,20]. The free energy of the pol /DNA ligase I interaction is dominated by hydrophobic interactions and the association is ionic strength sensitive [6]. Binding can be measured with the 8 kDa domain of the pol  domain rather than the 31 kDa domain. Finally, DNA pol  association with
324
H.T. Idriss et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 321–324
XRCC1 appears to enable DNA ligase III to replace the functions of DNA ligase I [20]. In summary, DNA pol  is very well suited for its role in AP site BER for several reasons: (1) its DNA synthesis specificity for short gaps; (2) its associated dRlyase activity; and (3) its ability to associate with other BER enzymes including DNA ligase I, AP endonuclease and XRCC1-DNA ligase III, among others [21,22].
Acknowledgements We thank Dr. William A. Beard, NIEHS, for supplying the figures. References [1] C.A. Brautigam, T.A. Steitz, Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes, Curr. Opin. Struct. Biol. 8 (1998) 54–63. [2] P.M.J. Burgers, Eukaryotic DNA polymerases in DNA replication and DNA repair, Chromosoma 107 (1998) 218– 227. [3] S. Waga, B. Stillman, The DNA replication fork in eukaryotic cells, Ann. Rev. Biochem. 67 (1998) 721–751. [4] M. Foiani, G. Lucchini, P. Plevani, The DNA polymerase alpha primase complex couples DNA replication, cell-cycle progression and DNA-damage response, Trends Biochem. Sci. 22 (1997) 424–427. [5] T.A. Steitz, DNA polymerases: structural diversity and common mechanisms, J. Biol. Chem. 274 (1999) 17395– 17398. [6] R. Prasad, et al., Functional analysis of the amino-terminus 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. [7] Y. Matsumoto, K. Kim, Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair, Science 269 (1995) 699–702. [8] Y. Matsumoto, K. Kim, DNA polymerase beta releases 5 -terminal deoxyribose-phosphate residues from incised abasic sites, J. Cell. Biochem. (1995) 288–288.
[9] H. Pelletier, M.R. Sawaya, A. Kumar, S.H. Wilson, J. Kraut, Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and Ddctp, Science 264 (1994) 1891– 1903. [10] 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. [11] A. Klungland, T. Lindahl, Second pathway for completion of human DNA base excision repair: reconstitution with purified proteins and requirement for DNase IV (Fen1), EMBOJ 16 (1997) 3341–3348. [12] M. Stucki, et al., Mammalian base excision repair by DNA polymerases delta and epsilon, Oncogene 17 (1998) 835–843. [13] G.L. Dianov, R. Prasad, S.H. Wilson, V.A. Bohr, Role of DNA polymerase beta in the excision step of long-patch mammalian base excision repair, J. Biol. Chem. 274 (1999) 13741–13743. [14] A.W. Plug, C.A. Clairmont, E. Sapi, T. Ashley, J.B. Sweasy, Evidence for a role for DNA polymerase beta in mammalian meiosis, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 1327–1331. [15] S.H. Wilson, Mammalian base excision repair and DNA polymerase beta, Mut. Res. DNA Repair 407 (1998) 203–215. [16] S.S. Parikh, C.D. Mol, J.A. Tainer, Base excision repair enzyme family portrait: integrating the structure and chemistry of an entire DNA repair pathway, Structure 5 (1997) 1543– 1550. [17] T.E. Wilson, M.R. Lieber, Efficient processing of DNA ends during yeast non-homologous end joining: evidence for a DNA polymerase beta (POL4)-dependent pathway, J. Biol. Chem. 274 (1999) 23599–23609. [18] R. Prasad, et al., Specific interaction of DNA polymerase beta and DNA ligase I in a multi-protein base excision repair complex from bovine testis, J. Biol. Chem. 271 (1996) 16000– 16007. [19] R.K. Singhal, S.H. Wilson, Short gap-filling synthesis by DNA polymerase beta is processive, J. Biol. Chem. 268 (1993) 15906–15911. [20] T. Lindahl, Recognition and processing of damaged DNA, J. Cell Sci. 19 (1995) 73–77. [21] J.K. Horton, R. Prasad, E.W. Hou, S.H. Wilson, Protection against methylation-induced cytotoxicity by DNA polymerase -dependent long patch base excision repair, J. Biol. Chem. 275 (2000) 2211–2218. [22] S.H. Wilson, T.A. Kunkel, Passing the Baton in Base Excision Repair, Nature Structural Biology 7 (2000) 176–178. [23] A. Nicholls, K.A. Sharp, B. Honig, Protein folding and association: insight from the interfacial and thermodynamic properties of hydrocarbons, Proteins 11 (1991) 281–296.
Molecules in focus
DNA polymerase  Haitham T. Idriss a,∗ , Osama Al-Assar b , Samuel H. Wilson a a
Laboratory of Structural Biology, NIEHS, NIH, Research Triangle Park, North Carolina, NC 29907, USA b Biomedical Research Centre, Medical School, University of Dundee, Ninewells Hospital Dundee DD1 9SY, Dundee, UK Received 20 July 2001; accepted 4 October 2001
Abstract Mammalian DNA polymerase (-pol) is a single polypeptide chain enzyme of 39 kDa. -pol has enzymatic activities appropriate for roles in base excision repair and other DNA metabolism events involving gap-filling DNA synthesis. Many crystal structures of -pol complexed with dNTP and DNA substrates have been solved, and mouse fibroblast cell lines deleted in the -pol gene have been examined. These approaches have enhanced our understanding of structural and functional aspects of -pol’s role in protecting genomic DNA. © 2002 Published by Elsevier Science Ltd. Keywords: DNA synthesis; DNA polymerase ; Base excision repair
1. Introduction Living cells face the tremendous task of maintaining an intact genome during their life span. This is necessary for cells to function in a complex environment, divide at the required time and die when appropriate. DNA synthesis is therefore necessary to duplicate the genome before cell division commences. DNA synthesis is also necessary during DNA repair. DNA replication is carried out by enzymes called DNA polymerases (DNA pols) [1]. The number of DNA pols has increased since the initial discovery of DNA pol ␣ in eukaryotic cells in 1957. In the early 1970s, DNA pol  and ␥ were discovered. This led to the immediate assumption that DNA pol ␣,  and ␥ are involved in DNA replication, repair and mitochon∗ Corresponding author. Tel.: +1-919-1-541-2644; fax: +1-919-1-541-4724. E-mail address: [email protected] (H.T. Idriss).
drial DNA replication, respectively. The discovery of DNA pol ␦ and ε during the 1980s complicated this interpretation. It was later suggested that DNA pols may serve more than one function [2]. DNA pols often have complex polypeptide structures (Fig. 1). In addition to the DNA polymerising function, which is often associated with a proofreading 3 → 5 exonuclease activity, DNA pols assume other important roles, such as DNA primase activity. Other functions include the ability to facilitate DNA polymerase interaction with other proteins involved in checkpoint function, cell cycle control, and DNA replication or repair [3]. DNA pols appear to possess a common catalytic active site. A two-metal ion catalysed mechanism guarantees the incorporation of the appropriate deoxyribonuleoside triphosphate base. However, DNA pols differ in various aspects of their structural architecture and it can be concluded that the active site of the DNA pol is conserved, whereas, the structure of
1357-2725/02/$ – see front matter © 2002 Published by Elsevier Science Ltd. PII: S 1 3 5 7 - 2 7 2 5 ( 0 1 ) 0 0 1 3 1 - 5
322
H.T. Idriss et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 321–324
Fig. 1. Genomic structure of DNA polymerase , illustrating the major domains in the protein molecule.
Fig. 2. Crystallographic structure for DNA polymerase  complexed with gapped DNA substrate. Image prepared using GRASP [23].
H.T. Idriss et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 321–324
the surface of the molecules might differ considerably [4,5]. 2. DNA polymerase  structure Pol  is a 39 kDa single chain polypeptide comprising 335 amino acid residues (Fig. 1). Experimental studies have shown that the enzyme is folded into distinct domains each associated with a specific functional activity. The amino-terminus (8 kDa) is connected to the polymerase domain (31 kDa) by a protease sensitive hinge region [6] Fig. 2. The 8 kDa domain has a lyase activity that removes the 5-deoxyribose phosphate generated after incision by an apurinic/apyrimidinic (AP) endonulcease during base excision repair (BER) [7,8]. The nucleotidyl transferase reaction is catalysed by the 31 kDa polymerase domain. In addition to the significant movement of the amino-terminus domain upon binding gapped DNA, the C-terminus is observed to ‘close’ around the correct incoming dNTP and its complementary template base [9,10]. 3. Roles of DNA pol  DNA pol  has been shown to be primarily involved in DNA repair. There are two biochemical pathways of BER in mammalian cells: short-patch (single nucleotide replacement) and long-patch BER (multi-nucleotide replacement) [4]. Experimental evidence indicates that DNA pol  is involved in short-patch BER. In addition, DNA pol ␦ or ε are probably involved in PCNA-dependent, long-patch BER with gaps of 2–13 nucleotides [11,12]. In this pathway, the flap endonuclease 1 (Fen1) is needed to cleave a reaction intermediate generated by template strand displacement during gap filling. A variation of the long-patch BER pathway with gap lengths of 2–6 nucleotides has also been characterised in mammalian cells. This Fen1 and PCNA-dependent pathway can be reconstituted with DNA pol ␦ and DNA pol ε. However, lack of DNA pol  in DNA pol  deficient cells or in the presence of neutralising antibody, causes a reduction in DNA repair activity. This strongly suggests a role for DNA pol  in this pathway in vivo [13].
323
Another role for DNA pol  is in meiosis and double strand break repair. DNA pol  has been implicated in the meiotic events associated with synapsis and recombination [14]. In addition, a 67 kDa homologue of mammalian DNA pol , encoded by the non-essential POL4 gene of S. cerevisiae, has recently been implicated in double strand break repair through a non-homologous end joining mechanism [15]. 4. DNA Pol  and BER Recent evidence has confirmed a role for DNA pol  in mammalian AP site BER. The working model for the short-patch BER pathway is as follows [16,17]. The glycosidic bond linking the damaged base and deoxyribose is cleaved either spontaneously or by a DNA glycosylase activity removing the inappropriate base to generate an abasic or AP site in double stranded DNA. The backbone of the phosphodiester of the AP site is cleaved 5 to the sugar moiety by AP endonuclease. This leaves a 3 -hydorxyl group and a deoxyribose phosphate group at the 5 -terminus. Subsequently, excision of the deoxyribose phosphate group is catalysed by 2-deoxyribose-5-phosphate lyase, an activity which is intrinsic to the amino-terminus of the DNA pol . The gap is filled by DNA pol  and DNA ligase seals the resulting nick. In addition to its dRlyase catalytic function, the 8 kDa N-terminus carries single strand DNA binding activity. The DNA binding specificity directs the enzyme to short gaps possessing a 5 -phosphate at the margin of the gap [18]. If the 5 -phosphate containing gap is one to six nucleotides, the enzyme binds and performs DNA synthesis in a processive fashion to completely fill the gap. However, if the gap size is larger than six nucleotides, the enzyme binds at the 5 margin of the gap but performs no DNA synthesis [19]. Therefore, processive DNA synthesis is coordinated by the 8 kDa domain of DNA pol  for a gap of six nucleotides or less. Several studies have shown DNA pol  to be closely associated with DNA ligase [18,20]. The free energy of the pol /DNA ligase I interaction is dominated by hydrophobic interactions and the association is ionic strength sensitive [6]. Binding can be measured with the 8 kDa domain of the pol  domain rather than the 31 kDa domain. Finally, DNA pol  association with
324
H.T. Idriss et al. / The International Journal of Biochemistry & Cell Biology 34 (2002) 321–324
XRCC1 appears to enable DNA ligase III to replace the functions of DNA ligase I [20]. In summary, DNA pol  is very well suited for its role in AP site BER for several reasons: (1) its DNA synthesis specificity for short gaps; (2) its associated dRlyase activity; and (3) its ability to associate with other BER enzymes including DNA ligase I, AP endonuclease and XRCC1-DNA ligase III, among others [21,22].
Acknowledgements We thank Dr. William A. Beard, NIEHS, for supplying the figures. References [1] C.A. Brautigam, T.A. Steitz, Structural and functional insights provided by crystal structures of DNA polymerases and their substrate complexes, Curr. Opin. Struct. Biol. 8 (1998) 54–63. [2] P.M.J. Burgers, Eukaryotic DNA polymerases in DNA replication and DNA repair, Chromosoma 107 (1998) 218– 227. [3] S. Waga, B. Stillman, The DNA replication fork in eukaryotic cells, Ann. Rev. Biochem. 67 (1998) 721–751. [4] M. Foiani, G. Lucchini, P. Plevani, The DNA polymerase alpha primase complex couples DNA replication, cell-cycle progression and DNA-damage response, Trends Biochem. Sci. 22 (1997) 424–427. [5] T.A. Steitz, DNA polymerases: structural diversity and common mechanisms, J. Biol. Chem. 274 (1999) 17395– 17398. [6] R. Prasad, et al., Functional analysis of the amino-terminus 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. [7] Y. Matsumoto, K. Kim, Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair, Science 269 (1995) 699–702. [8] Y. Matsumoto, K. Kim, DNA polymerase beta releases 5 -terminal deoxyribose-phosphate residues from incised abasic sites, J. Cell. Biochem. (1995) 288–288.
[9] H. Pelletier, M.R. Sawaya, A. Kumar, S.H. Wilson, J. Kraut, Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and Ddctp, Science 264 (1994) 1891– 1903. [10] 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. [11] A. Klungland, T. Lindahl, Second pathway for completion of human DNA base excision repair: reconstitution with purified proteins and requirement for DNase IV (Fen1), EMBOJ 16 (1997) 3341–3348. [12] M. Stucki, et al., Mammalian base excision repair by DNA polymerases delta and epsilon, Oncogene 17 (1998) 835–843. [13] G.L. Dianov, R. Prasad, S.H. Wilson, V.A. Bohr, Role of DNA polymerase beta in the excision step of long-patch mammalian base excision repair, J. Biol. Chem. 274 (1999) 13741–13743. [14] A.W. Plug, C.A. Clairmont, E. Sapi, T. Ashley, J.B. Sweasy, Evidence for a role for DNA polymerase beta in mammalian meiosis, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 1327–1331. [15] S.H. Wilson, Mammalian base excision repair and DNA polymerase beta, Mut. Res. DNA Repair 407 (1998) 203–215. [16] S.S. Parikh, C.D. Mol, J.A. Tainer, Base excision repair enzyme family portrait: integrating the structure and chemistry of an entire DNA repair pathway, Structure 5 (1997) 1543– 1550. [17] T.E. Wilson, M.R. Lieber, Efficient processing of DNA ends during yeast non-homologous end joining: evidence for a DNA polymerase beta (POL4)-dependent pathway, J. Biol. Chem. 274 (1999) 23599–23609. [18] R. Prasad, et al., Specific interaction of DNA polymerase beta and DNA ligase I in a multi-protein base excision repair complex from bovine testis, J. Biol. Chem. 271 (1996) 16000– 16007. [19] R.K. Singhal, S.H. Wilson, Short gap-filling synthesis by DNA polymerase beta is processive, J. Biol. Chem. 268 (1993) 15906–15911. [20] T. Lindahl, Recognition and processing of damaged DNA, J. Cell Sci. 19 (1995) 73–77. [21] J.K. Horton, R. Prasad, E.W. Hou, S.H. Wilson, Protection against methylation-induced cytotoxicity by DNA polymerase -dependent long patch base excision repair, J. Biol. Chem. 275 (2000) 2211–2218. [22] S.H. Wilson, T.A. Kunkel, Passing the Baton in Base Excision Repair, Nature Structural Biology 7 (2000) 176–178. [23] A. Nicholls, K.A. Sharp, B. Honig, Protein folding and association: insight from the interfacial and thermodynamic properties of hydrocarbons, Proteins 11 (1991) 281–296.