Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

Department of Biology, New York University, 100 Washington Square East, 1009 Silver Center, New York, NY 10003, USA. <>
Trends in Biochemical Sciences (Impact Factor: 11.23). 06/2008; 33(5):209-19. DOI: 10.1016/j.tibs.2008.02.004
Source: PubMed


When a high-fidelity DNA polymerase encounters certain DNA-damage sites, its progress can be stalled and one or more lesion-bypass polymerases are recruited to transit the lesion. Here, we consider two representative types of lesions: (i) 7,8-dihydro-8-oxoguanine (8-oxoG), a small, highly prevalent lesion caused by oxidative damage; and (ii) bulky lesions derived from the environmental pre-carcinogen benzo[a]pyrene, in the high-fidelity DNA polymerase Bacillus fragment (BF) from Bacillus stearothermophilus and in the lesion-bypass DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. The tight fit of the BF polymerase around the nascent base pair contrasts with the more spacious, solvent-exposed active site of Dpo4, and these differences in architecture result in distinctions in their respective functions: one-step versus stepwise polymerase translocation, mutagenic versus accurate bypass of 8-oxoG, and polymerase stalling versus mutagenic bypass at bulky benzo[a]pyrene-derived lesions.

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Available from: Olga Rechkoblit, Jun 25, 2015
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    • "Another major structural difference between high-fidelity and Y-family polymerases is in the active site. To accommodate bulky DNA lesions or distorted DNA structures, they have a wider active site that makes fewer contacts with its substrate (12). However, the catalytic core remains highly conserved among the Y-family polymerases and presents a tricarboxylate moiety akin to polymerases from other families. "
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    ABSTRACT: Y-family DNA polymerases play a crucial role in translesion DNA synthesis. Here, we have characterized the binding kinetics and conformational dynamics of the Y-family polymerase Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) using single-molecule fluorescence. We find that in the absence of dNTPs, the binary complex shuttles between two different conformations within ∼1 s. These data are consistent with prior crystal structures in which the nucleotide binding site is either occupied by the terminal base pair (preinsertion conformation) or empty following Dpo4 translocation by 1 base pair (insertion conformation). Most interestingly, on dNTP binding, only the insertion conformation is observed and the correct dNTP stabilizes this complex compared with the binary complex, whereas incorrect dNTPs destabilize it. However, if the n+1 template base is complementary to the incoming dNTP, a structure consistent with a misaligned template conformation is observed, in which the template base at the n position loops out. This structure provides evidence for a Dpo4 mutagenesis pathway involving a transient misalignment mechanism.
    Nucleic Acids Research 11/2013; 42(4). DOI:10.1093/nar/gkt1149 · 9.11 Impact Factor
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    • "A DNAP appropriate for DNA recording of environmental signals should ideally have a wide dynamic range of misincorporation rates and be active at mesophilic temperatures. Dpo4 (Sulfolobus solfataricus) [12] is a member of the Y-family of polymerases [13], [14], which are implicated in translesion bypass [15] and somatic hypermutation [16] and have high misincorporation rates. Klenow exo− is the D355A, E357A mutant [17] of the Klenow Fragment of the E. coli DNA Polymerase I [18], which lacks 3′–5′ proofreading activity, and, unlike most commercially available DNAPs, is compatible with the 37°C extension temperature used for the Y-family enzymes. "
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    • "The Y family translesion synthesis (TLS) DNA polymerases are generally low fidelity on undamaged DNA (1) and have functions which include, among others, to bypass distorting bulky DNA lesions (2–7) that primarily stall high fidelity replicative polymerases; one or more members of the Y family TLS polymerases may then replace the high fidelity polymerase to bypass the region of distortion (7–9). This bypass may be error-free or error-prone, depending on the nature of the lesion and the specific polymerase (1–3,7). "
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    ABSTRACT: Human DNA Pol κ is a polymerase enzyme, specialized for near error-free bypass of certain bulky chemical lesions to DNA that are derived from environmental carcinogens present in tobacco smoke, automobile exhaust and cooked food. By employing ab initio QM/MM-MD (Quantum Mechanics/Molecular Mechanics-Molecular Dynamics) simulations with umbrella sampling, we have determined the entire free energy profile of the nucleotidyl transfer reaction catalyzed by Pol κ and provided detailed mechanistic insights. Our results show that a variant of the Water Mediated and Substrate Assisted (WMSA) mechanism that we previously deduced for Dpo4 and T7 DNA polymerases is preferred for Pol κ as well, suggesting its broad applicability. The hydrogen on the 3'-OH primer terminus is transferred through crystal and solvent waters to the γ-phosphate of the dNTP, followed by the associative nucleotidyl transfer reaction; this is facilitated by a proton transfer from the γ-phosphate to the α,β-bridging oxygen as pyrophosphate leaves, to neutralize the evolving negative charge. MD simulations show that the near error-free incorporation of dCTP opposite the major benzo[a]pyrene-derived dG lesion is compatible with the WMSA mechanism, allowing for an essentially undisturbed pentacovalent phosphorane transition state, and explaining the bypass of this lesion with little mutation by Pol κ.
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