A review of recent experiments on step-to-step "hand-off" of the DNA intermediates in mammalian base excision repair pathways.

The current "working model" for mammalian base excision repair involves two sub-pathways termed single-nucleotide base excision repair and long patch base excision repair that are distinguished by their repair patch sizes and the enzymes/co-factors involved. These base excision repair sub-pathways are designed to sequester the various DNA intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the base excision repair intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In this review, we explore the question of whether there is an actual step-to-step "hand-off" of the DNA intermediates during base excision repair in vitro. The results show that when base excision repair enzymes are pre-bound to the initial single-nucleotide base excision repair intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase beta and then to DNA ligase. In the long patch base excision repair sub-pathway, where the 5'-end of the incised strand is blocked, the intermediate after polymerase beta gap filling is not channeled from polymerase beta to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.

DNA lesion bypass polymerases open up.

Structures of catalytic fragments of two DNA lesion bypass DNA polymerases, yeast DNA polymerase eta and an archeon DinB homolog, have recently been solved. These structures share several common architectural and structural features observed in other DNA polymerases, including a hand-like architecture with fingers, palm, and thumb subdomains. The new structures provide the first structural insights into DNA lesion bypass. The fingers and thumb are smaller than those in other DNA polymerases. Modeled substrates suggest that the fingers in the vicinity of the incoming nucleotide is closed, a conformation not previously observed for an unliganded polymerase. However, the template binding pocket appears to be more open, indicating that for DNA polymerase eta, a covalently linked thymine-thymine dimer could be accommodated.

DNA structure and aspartate 276 influence nucleotide binding to human DNA polymerase beta. Implication for the identity of the rate-limiting conformational change.

Structures of DNA polymerase (pol) beta bound to single-nucleotide gapped DNA had revealed that the lyase and pol domains form a "doughnut-shaped" structure altering the dNTP binding pocket in a fashion that is not observed when bound to non-gapped DNA. We have investigated dNTP binding to pol beta-DNA complexes employing steady-state and pre-steady-state kinetics. Although pol beta has a kinetic scheme similar to other DNA polymerases, polymerization by pol beta is limited by at least two partially rate-limiting steps: a conformational change after dNTP ground-state binding and product release. The equilibrium binding constant, K(d)((dNTP)), decreased and the insertion efficiency increased with a one-nucleotide gapped DNA substrate, as compared with non-gapped DNA. Valine substitution for Asp(276), which interacts with the base of the incoming nucleotide, increased the binding affinity for the incoming nucleotide indicating that the negative charge contributed by Asp(276) weakens binding and that an interaction between residue 276 with the incoming nucleotide occurs during ground-state binding. Since the interaction between Asp(276) and the nascent base pair is observed only in the "closed" conformation of pol beta, the increased free energy in ground-state binding for the mutant suggests that the subsequent rate-limiting conformational change is not the "open" to "closed" structural transition, but instead is triggered in the closed pol conformation.

Structural design of a eukaryotic DNA repair polymerase: DNA polymerase beta.

DNA polymerase beta, the smallest eukaryotic DNA polymerase, is designed to synthesize DNA in short DNA gaps during DNA repair. It is composed of two specialized domains that contribute essential enzymatic activities to base excision repair (BER). Its amino-terminal domain possesses a lyase activity necessary to remove the 5'-deoxyribose phosphate (dRP) intermediate generated during BER. Removal of the dRP moiety is often the rate-limiting step during BER. Failure to remove this group may initiate alternate BER pathways. The larger polymerase domain has nucleotidyl transferase activity. This domain has a modular organization with sub-domains that bind duplex DNA, catalytic metals, and the correct nucleoside triphosphate in a template-dependent manner. X-ray crystal structures of DNA polymerase beta, with and without bound substrates, has inferred that domain, sub-domain, and substrate conformational changes occur upon ligand binding. Many of these conformational changes are distinct from those observed in structures of other DNA polymerases. This review will examine the structural aspects of DNA polymerase beta that facilitate its role in BER.

Minor groove interactions at the DNA polymerase beta active site modulate single-base deletion error rates.

The structures of open and closed conformations of DNA polymerase beta (pol beta) suggests that the rate of single-nucleotide deletions during synthesis may be modulated by interactions in the DNA minor groove that align the templating base with the incoming dNTP. To test this hypothesis, we measured the single-base deletion error rates of wild-type pol beta and lysine and alanine mutants of Arg(283), whose side chain interacts with the minor groove edge of the templating nucleotide at the active site. The error rates of both mutant enzymes are increased >100-fold relative to wild-type pol beta. Template engineering experiments performed to distinguish among three possible models for deletion formation suggest that most deletions in repetitive sequences by pol beta initiate by strand slippage. However, pol beta also generates deletions by a different mechanism that is strongly enhanced by the substitutions at Arg(283). Analysis of error specificity suggests that this mechanism involves nucleotide misinsertion followed by primer relocation, creating a misaligned intermediate. The structure of pol beta bound to non-gapped DNA also indicates that the templating nucleotide and its downstream neighbor are out of register in the open conformation and this could facilitate misalignment (dNTP or primer terminus) with the next template base.

Vertical-scanning mutagenesis of a critical tryptophan in the "minor groove binding track" of HIV-1 reverse transcriptase. Major groove DNA adducts identify specific protein interactions in the minor groove.

Biochemical and molecular modeling studies of human immunodeficiency virus type 1 reverse transcriptase (RT) have revealed that a structural element, the minor groove binding track (MGBT), is important for both replication frameshift fidelity and processivity. The MGBT interactions occur in the DNA minor groove from the second through sixth base pair from the primer 3'-terminus where the DNA undergoes a structural transition from A-like to B-form DNA. Alanine-scanning mutagenesis had previously demonstrated that Gly(262) and Trp(266) of the MGBT contributes important DNA interactions. To probe the molecular interactions occurring in this critical region, eight mutants of RT were studied in which alternate residues were substituted for Trp(266). These enzymes were characterized in primer extension assays in which the template DNA was adducted at a single adenine by either R- or S-enantiomers of styrene oxide. These lesions failed to block DNA polymerization by wild-type RT, yet the Trp(266) mutants and an alanine mutant of Gly(262) terminated synthesis on styrene oxide-adducted templates. Significantly, the sites of termination occurred primarily 1 and 3 bases following adduct bypass, when the lesion was positioned in the major groove of the template-primer stem. These results indicate that residue 266 serves as a "protein sensor" of altered minor groove interactions and identifies which base pair interactions are altered by these lesions. In addition, the major groove lesion must alter important structural transitions in the template-primer stem, such as minor groove widening, that allow RT access to the minor groove.

Molecular cloning and high-level expression of human polymerase beta cDNA and comparison of the purified recombinant human and rat enzymes.

The cDNA encoding the human polymerase beta from HeLa cells was PCR amplified and cloned, and its nucleotide sequence determined. The DNA sequence is identical to the polymerase beta cDNA sequence from Tera-2 cells. Three expression strategies were employed that were designed to maximize translation initiation of the polymerase beta mRNA in Escherichia coli and all yielded a high level of human polymerase beta. The recombinant protein was purified and its properties were compared with those of the recombinant rat enzyme. The domain structure and kinetic parameters (k(cat) and K(m)) were nearly identical. A mouse IgG monoclonal antibody to the rat enzyme (mAb-10S) was approximately 10-fold less reactive with the human enzyme than with the rat enzyme as determined by ELISA.

Uniquely altered DNA replication fidelity conferred by an amino acid change in the nucleotide binding pocket of human immunodeficiency virus type 1 reverse transcriptase.

Arginine 72 in human immunodeficiency virus type 1 reverse transcriptase (RT), a highly conserved residue among retroviral polymerases and telomerases, forms part of the binding pocket for the nascent base pair. We show here that replacement of Arg(72) by alanine strongly alters fidelity in a highly unusual manner. R72A reverse transcriptase is a frameshift and base substitution antimutator polymerase whose increased fidelity results both from increased nucleotide selectivity and from a decreased ability to extend mismatched primer termini. Thus, Arg(72)-substrate interactions in wild-type human immunodeficiency virus type 1 RT can stabilize incorrect nucleotides allowing misinsertion and promoting extension of mismatched and perhaps misaligned template-primers. In contrast to the higher fidelity at most sites, R72A RT is highly error-prone for misincorporations opposite template T in the sequence context: 5'-CTGG. Surprisingly, this results mostly from a 1200-fold increase in the apparent K(m) for correct dAMP incorporation. Thus, Arg(72) interactions with substrate are critical for the stability of the correct T.dAMP base pair when the 5'-CTGG sequence is present in the binding pocket for the nascent base pair. Collectively, the data show that a mutant polymerase may yield higher than normal average replication fidelity, yet paradoxically place specific sequences at very high risk of mutation.

Residues in the alphaH and alphaI helices of the HIV-1 reverse transcriptase thumb subdomain required for the specificity of RNase H-catalyzed removal of the polypurine tract primer.

During retrovirus replication, reverse transcriptase (RT) must specifically interact with the polypurine tract (PPT) to generate and subsequently remove the RNA primer for plus-strand DNA synthesis. We have investigated the role that human immunodeficiency virus-1 RT residues in the alphaH and alphaI helices in the thumb subdomain play in specific RNase H cleavage at the 3'-end of the PPT; an in vitro assay modeling the primer removal step was used. Analysis of alanine-scanning mutants revealed that a subgroup exhibits an unusual phenotype in which the PPT is cleaved up to seven bases from its 3'-end. Further analysis of alphaH mutants (G262A, K263A, N265A, and W266A) with changes in residues in or near a structural motif known as the minor groove binding track showed that the RNase H activity of these mutants is more dramatically affected with PPT substrates than with non-PPT substrates. Vertical scan mutants at position 266 were all defective in specific RNase H cleavage, consistent with conservation of tryptophan at this position among lentiviral RTs. Our results indicate that residues in the thumb subdomain and the minor groove binding track in particular, are crucial for unique interactions between RT and the PPT required for correct positioning and precise RNase H cleavage.

Base substitution specificity of DNA polymerase beta depends on interactions in the DNA minor groove.

To examine the hypothesis that interactions between a DNA polymerase and the DNA minor groove are critical for accurate DNA synthesis, we studied the fidelity of DNA polymerase beta mutants at residue Arg(283), where arginine, which interacts with the minor groove at the active site, is replaced by alanine or lysine. Alanine substitution, removing minor groove interactions, strongly reduces polymerase selectivity for all single-base mispairs examined. In contrast, the lysine substitution, which retains significant interactions with the minor groove, has wild-type-like selectivity for T.dGMP and A.dGMP mispairs but reduced selectivity for T.dCMP and A.dCMP mispairs. Examination of DNA crystal structures of these four mispairs indicates that the two mispairs excluded by the lysine mutant have an atom (N2) in an unfavorable position in the minor groove, while the two mispairs permitted by the lysine mutant do not. These results suggest that unfavorable interactions between an active site amino acid side chain and mispair-specific atoms in the minor groove contribute to DNA polymerase specificity.

The fidelity of DNA polymerase beta during distributive and processive DNA synthesis.

During base excision repair, DNA polymerase beta fills 1-6-nucleotide gaps processively, reflecting a contribution of both its 8- and 31-kDa domains to DNA binding. Here we report the fidelity of pol beta during synthesis to fill gaps of 1, 5, 6, or >300 nucleotides. Error rates during distributive synthesis by recombinant rat and human polymerase (pol) beta with a 390-base gap are similar to each other and to previous values with pol beta purified from tissues. The base substitution fidelity of human pol beta when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but "closely-spaced" substitutions are produced at a rate at least 60-fold higher than for distributive synthesis. Base substitution fidelity when filling a 1-nucleotide gap is higher than when filling a 5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the dNTP binding pocket and/or a difference in base stacking or DNA structure imposed by pol beta. Nonetheless, 1-nucleotide gap filling is inaccurate, even generating complex substitution-addition errors. Finally, the single-base deletion error rate during processive synthesis to fill a 6-nucleotide gap is indistinguishable from that of distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism of processivity by pol beta does not allow the enzyme to suppress template misalignments.

Vertical-scanning mutagenesis of a critical tryptophan in the minor groove binding track of HIV-1 reverse transcriptase. Molecular nature of polymerase-nucleic acid interactions.

While sequence-specific DNA-binding proteins interact predominantly in the DNA major groove, DNA polymerases bind DNA through interactions in the minor groove that are sequence nonspecific. Through functional analyses of alanine-substituted mutant enzymes that were guided by molecular dynamics modeling of the human immunodeficiency virus type 1-reverse transcriptase and DNA complex, we previously identified a structural element in reverse transcriptase, the minor groove binding track (MGBT). The MGBT is comprised of five residues (Ile94, Gln258, Gly262, Trp266, and Gln269) which interact 2-6 base pairs upstream from the polymerase active site in the DNA minor groove and are important in DNA binding, processivity, and frameshift fidelity. These residues do not contribute equally; functional analysis of alanine mutants suggests that Trp266 contributes the most to binding. To define the molecular interactions between Trp266 and the DNA minor groove, we have analyzed the properties of eight mutants, each with an alternate side chain at this position. A refined molecular dynamics model was used to calculate relative binding free energies based on apolar surface area buried upon complex formation. In general, there was a strong correlation between the relative calculated binding free energies for the alternate residue 266 side chains and the magnitude of the change in the properties which reflect template-primer interactions (template-primer dissociation rate constant, Ki,AZTTP, processivity, and frameshift fidelity). This correlation suggests that hydrophobic interactions make a major contribution to the stability of the polymerase-DNA complex. Additionally, tyrosine and arginine substitutions resulted in mutant enzymes with DNA binding properties better than predicted by buried surface area alone, suggesting that hydrogen bonding could also play a role in DNA binding at this position.

Mammalian abasic site base excision repair. Identification of the reaction sequence and rate-determining steps.

Base excision repair (BER) is one of the cellular defense mechanisms repairing damage to nucleoside 5'-monophosphate residues in genomic DNA. This repair pathway is initiated by spontaneous or enzymatic N-glycosidic bond cleavage creating an abasic or apurinic-apyrimidinic (AP) site in double-stranded DNA. Class II AP endonuclease, deoxyribonucleotide phosphate (dRP) lyase, DNA synthesis, and DNA ligase activities complete repair of the AP site. In mammalian cell nuclear extract, BER can be mediated by a macromolecular complex containing DNA polymerase beta (beta-pol) and DNA ligase I. These two enzymes are capable of contributing the latter three of the four BER enzymatic activities. In the present study, we found that AP site BER can be reconstituted in vitro using the following purified human proteins: AP endonuclease, beta-pol, and DNA ligase I. Examination of the individual enzymatic steps in BER allowed us to identify an ordered reaction pathway: subsequent to 5' "nicking" of the AP site-containing DNA strand by AP endonuclease, beta-pol performs DNA synthesis prior to removal of the 5'-dRP moiety in the gap. Removal of the dRP flap is strictly required for DNA ligase I to seal the resulting nick. Additionally, the catalytic rate of the reconstituted BER system and the individual enzymatic activities was measured. The reconstituted BER system performs repair of AP site DNA at a rate that is slower than the respective rates of AP endonuclease, DNA synthesis, and ligation, suggesting that these steps are not rate-determining in the overall reconstituted BER system. Instead, the rate-limiting step in the reconstituted system was found to be removal of dRP (i.e. dRP lyase), catalyzed by the amino-terminal domain of beta-pol. This work is the first to measure the rate of BER in an in vitro reaction. The potential significance of the dRP-containing intermediate in the regulation of BER is discussed.

Functional analysis of the amino-terminal 8-kDa domain of DNA polymerase beta as revealed by site-directed mutagenesis. DNA binding and 5'-deoxyribose phosphate lyase activities.

The amino-terminal 8-kDa domain of DNA polymerase beta functions in binding single-stranded DNA (ssDNA), recognition of a 5'-phosphate in gapped DNA structures, and as a 5'-deoxyribose phosphate (dRP) lyase. NMR and x-ray crystal structures of this domain have suggested several residues that may interact with ssDNA or play a role in the dRP lyase reaction. Nine of these residues were altered by site-directed mutagenesis. Each mutant was expressed in Escherichia coli, and the recombinant protein was purified to near homogeneity. CD spectra of these mutant proteins indicated that the alteration did not adversely affect the global protein structure. Single-stranded DNA binding was probed by photochemical cross-linking to oligo(dT)16. Several mutants (F25W, K35A, K60A, and K68A) were impaired in ssDNA binding activity, whereas other mutants (H34G, E71Q, K72A, E75A, and K84A) retained near wild-type binding activity. The 5'-phosphate recognition activity of these mutants was examined by UV cross-linking to a 5-nucleotide gap DNA where the 5' terminus in the gap was either phosphorylated or unphosphorylated. The results indicate that Lys35 is involved in 5'-phosphate recognition of DNA polymerase beta. Finally, the dRP lyase activity of these mutants was evaluated using a preincised apurinic/apyrimidinic DNA. Alanine mutants of Lys35 and Lys60 are significantly reduced in dRP lyase activity, consistent with the lower ssDNA binding activity. More importantly, alanine substitution for Lys72 resulted in a greater than 90% loss of dRP lyase activity, without affecting DNA binding. Alanine mutants of Lys68 and Lys84 had wild-type dRP lyase activity. The triple alanine mutant, K35A/K68A/K72A, was devoid of dRP lyase activity, suggesting that the effects of the alanine substitution at Lys72 and Lys35 were additive. The results suggest that Lys72 is directly involved in formation of a covalent imino intermediate and are consistent with Lys72 as the predominant Schiff base nucleophile in the dRP lyase beta-elimination catalytic reaction.

Human DNA polymerase beta deoxyribose phosphate lyase. Substrate specificity and catalytic mechanism.

DNA polymerase beta (beta-pol) cleaves the sugar-phosphate bond 3' to an intact apurinic/apyrimidinic (AP) site (i.e. AP lyase activity). The same bond is cleaved even if the AP site has been previously 5'-incised by AP endonuclease, resulting in a 5' 2-deoxyribose 5-phosphate (i.e. dRP lyase activity). We characterized these lyase reactions by steady-state kinetics with the amino-terminal 8-kDa domain of beta-pol and with the entire 39-kDa polymerase. Steady-state kinetic analyses show that the Michaelis constants for both the dRP and AP lyase activities of beta-pol are similar. However, kcat is approximately 200-fold lower for the AP lyase activity on an intact AP site than for an AP endonuclease-preincised site. The 8-kDa domain was also less efficient with an intact AP site than on a preincised site. The full-length enzyme and the 8-kDa domain efficiently remove the 5' dRP from a preincised AP site in the absence of Mg2+, and the pH profiles of beta-pol and 8-kDa domain dRP lyase catalytic efficiency exhibit a broad alkaline pH optimum. An inhibitory effect of pyridoxal 5'-phosphate on the dRP lyase activity is consistent with involvement of a primary amine (Lys72) as the Schiff base nucleophile during lyase chemistry.

Interactions between DNA polymerase beta and the major covalent adduct of the carcinogen (+)-anti-benzo[a]pyrene diol epoxide with DNA at a primer-template junction.

A molecular dynamics simulation has been carried out with DNA polymerase beta (beta pol) complexed with a DNA primer-template. The templating guanine at the polymerase active site was covalently modified by the carcinogenic metabolite of benzo[a]pyrene, (+)-anti-benzo[a]pyrene diol epoxide, to form the major (+)-trans-anti-benzo[a]pyrene diol epoxide covalent adduct. Thus, the benzo[a]pyrenyl moiety (BP) is situated in the single-stranded template at the junction between double- and single-stranded DNA. The starting structure was based on the X-ray crystal structure of the rat beta pol primer-template and ddCTP complex [Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903]. During the simulation, the BP and its attached templating guanine rearrange to form a structure in which the BP is closer to parallel with the adjacent base pair. In addition, the templating attached guanine is displaced toward the major groove side and access to its Watson-Crick edge is partly obstructed. This structure is stabilized, in part, by new hydrogen bonds between the BP and beta pol Asn279 and Arg283. These residues are within hydrogen bonding distance to the incoming ddCTP and templating guanine, respectively, in the crystal structure of the beta pol ternary complex. Site-directed mutagenesis has confirmed their role in dNTP binding, discrimination, and catalytic efficiency [Beard, W. A., Osheroff, W. P., Prasad, R., Sawaya, M. R., Jaju, M., Wood, T. G., Kraut, J., Kunkel, T. A., and Wilson, S. H. (1996) J. Biol. Chem. 271, 12141-12144]. The predominant biological effect of the BP is DNA polymerase blockage. Consistent with this biological effect, the computed structure suggests the possibility that the BP's main deleterious impact on DNA synthesis might result at least in part from its specific interactions with key polymerase side chains. Moreover, relatively modest movement of BP and its attached guanine, with some concomitant enzyme motion, is necessary to relieve the obstruction and permit the observed rare incorporation of a dATP opposite the guanine lesion.

Subunits of human replication protein A are crosslinked by photoreactive primers synthesized by DNA polymerases.

Human replication protein A (huRPA) is a multisubunit protein which is involved in DNA replication, repair and recombination processes. It exists as a stable heterotrimer consisting of p70, p32 and p14 subunits. To understand the contribution of huRPA subunits to DNA binding we applied the photoaffinity labeling technique. The photoreactive oligonucleotide was synthesized in situ by DNA polymerases. 5-[N-(2-nitro-5-azidobenzoyl)-trans -3-aminopropenyl-1]deoxyuridine-5'-triphosphate (NABdUTP) was used as substrate for elongation of a radiolabeled primer logical ortemplate either by human DNA polymerase alpha primase (polalpha), human DNA polymerase beta (polbeta) or Klenow fragment of Escherichia coli DNA polymerase I (KF). The polymerase was incubated with NABdUTP and radiolabeled primer-template in the presence or absence of huRPA. The reaction mixtures were then irradiated with monochromatic UV light (315 nm) and the crosslinked products were separated by SDS-PAGE. The results clearly demonstrate crosslinking of the huRPA p70 and p32 subunits with DNA. The p70 subunit appears to bind to the single-stranded part of the DNA duplex, the p32 subunit locates near the 3'-end of the primer, while the p14 subunit locates relatively far from the 3'-end of the primer. This approach opens new possibilities for analysis of huRPA loading on DNA in the course of DNA replication and DNA repair.

Structural insights into DNA polymerase beta fidelity: hold tight if you want it right.

DNA polymerases must select and incorporate the correct deoxynucleoside 5'-triphosphate from a pool of structurally similar molecules. The structural and kinetic characterization of DNA polymerase beta indicates that this polymerase must stabilize the templating base to achieve efficient polymerization with high fidelity.