Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

Abstract

1-Nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dG AP ). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dG AP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dG AP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.Environmental pollutants have been shown to impact human health at the molecular level. One detrimental route is the modification of genomic DNA and nucleotides (1). If DNA lesions are not recognized and removed by the cellular DNA repair machinery, they will stall replicative DNA polymerases (2-8).To rescue DNA replication, cells employ lesion bypass DNA polymerases to traverse unrepaired lesions. Most of these enzymes belong to the Y-family of DNA polymerases. The Y-family enzymes possess relatively flexible and solvent-accessible active sites to accommodate bulky DNA lesions (9, 10). However, Y-family DNA polymerases catalyze DNA synthesis over undamaged DNA with low fidelity and poor processivity (6, 10 -12). The Y-family DNA polymerases have been identified in all three domains of life, e.g. four in humans (DNA polymerases , , , and Rev1), two in Escherichia coli (DNA polymerases IV and V), and one in Sulfolobus solfataricus (Dpo4). Because Dpo4 can be expressed in E. coli and purified with a high yield, it has been extensively studied in vitro as a prototype Y-family enzyme. Dpo4 catalyzes DNA synthesis on an undamaged DNA template with a fidelity of one error per 1,000 -10,000 nucleotide incorporations based on presteady-state kinetic analysis from 37 to 56°C (13-15). Dpo4 is capable of bypassing a myriad of DNA lesions including apurinic/apyrimidinic (abasic) sites (16 -19), 8-oxo-7,8-dihydro-2Ј-deoxyguanosine (20, 21), 1,N 2 -etheno(⑀)guanosine (22), cis-syn thymine-thymine dimer (23-...

Mechanistic Basis for the Bypass of a Bulky DNA Adduct Catalyzed by a Y-Family DNA Polymerase

“…The 25-mer accumulation was likely due to polymerase “slippage” via primer realignment at the dC-rich sequence of the 5′-termini of the damaged and undamaged 26-mer templates (Table 1). 41 …”

“…In contrast, similar DNA trap experiments with the undamaged DNA substrates and the damaged DNA substrates at two nonpause sites only exhibited monophasic kinetics (Table 4). As previously rationalized, DNA trap assay results for the bypass of an abasic site, 55 a cisplatin-d(GpG) adduct, 56 a dG AP lesion, 41 and an N -(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone adduct (dG C8- N -ABA ) 57 by Dpo4, the fast phase observed with 20/26-mer-dG 1,8 was due to the formation of a productive complex E·DNA n P which was quickly turned over to 21/26-mer-dG 1,8 once dCTP was bound. Notably, with 19-mer, 20-mer, or 21-mer as the primer, the k f is smaller with the damaged than with the undamaged DNA substrate.…”

“… a Values for K d ,DNA are from ref 41. b Damaged DNA refers to those with template 26-mer-dG 1,8 in Table 1. c Calculated as ( K d ,DNA ) damaged / ( K d ,DNA ) undamaged . …”

Structural Mechanism of Ribonucleotide Discrimination by a Y-Family DNA Polymerase

“…7 Interestingly, DNA polymerases pol β and pol λ from the X-family utilize the protein backbone for ribonucleotide discrimination rather than a specific steric gate residue. 10,37 Nevertheless, NTP clashing appears to be a general mechanism of all DNA polymerases.…”

“…2,3 Most DNA polymerases utilize bulky side-chain residues in their active sites to discriminate against NTPs. 4 The identity of this residue is typically glutamine for Afamily DNA polymerases 5,6 and tyrosine or phenylalanine for Y-and B-family DNA polymerases [7][8][9][10] (Fig. 1b).…”