Bypass of DNA-Protein Cross-links Conjugated to the 7-Deazaguanine Position of DNA by Translesion Synthesis Polymerases.

DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.

Structural and dynamic characterization of polymerase κ's minor groove lesion processing reveals how adduct topology impacts fidelity.

DNA lesion bypass polymerases process different lesions with varying fidelities, but the structural, dynamic, and mechanistic origins of this phenomenon remain poorly understood. Human DNA polymerase κ (Polκ), a member of the Y family of lesion bypass polymerases, is specialized to bypass bulky DNA minor groove lesions in a predominantly error-free manner, by housing them in its unique gap. We have investigated the role of the unique Polκ gap and N-clasp structural features in the fidelity of minor groove lesion processing with extensive molecular modeling and molecular dynamics simulations to pinpoint their functioning in lesion bypass. Here we consider the N(2)-dG covalent adduct derived from the carcinogenic aromatic amine, 2-acetylaminofluorene (dG-N(2)-AAF), that is produced via the combustion of kerosene and diesel fuel. Our simulations reveal how the spacious gap directionally accommodates the lesion aromatic ring system as it transits through the stages of incorporation of the predominant correct partner dCTP opposite the damaged guanine, with preservation of local active site organization for nucleotidyl transfer. Furthermore, flexibility in Polκ's N-clasp facilitates the significant misincorporation of dTTP opposite dG-N(2)-AAF via wobble pairing. Notably, we show that N-clasp flexibility depends on lesion topology, being markedly reduced in the case of the benzo[a]pyrene-derived major adduct to N(2)-dG, whose bypass by Polκ is nearly error-free. Thus, our studies reveal how Polκ's unique structural and dynamic properties can regulate its bypass fidelity of polycyclic aromatic lesions and how the fidelity is impacted by lesion structures.

Preferred WMSA catalytic mechanism of the nucleotidyl transfer reaction in human DNA polymerase κ elucidates error-free bypass of a bulky DNA lesion.

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,ß-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.

DNA cytosine methylation: structural and thermodynamic characterization of the epigenetic marking mechanism.

DNA cytosine methyltransferases regulate the expression of the genome through the precise epigenetic marking of certain cytosines with a methyl group, and aberrant methylation is a hallmark of human diseases including cancer. Targeting these enzymes for drug design is currently a high priority. We have utilized ab initio quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations to investigate extensively the reaction mechanism of the representative DNA methyltransferase HhaI (M.HhaI) from prokaryotes, whose overall mechanism is shared with the mammalian enzymes. We obtain for the first time full free energy profiles for the complete reaction, together with reaction dynamics in atomistic detail. Our results show an energetically preferred mechanism in which nucleophilic attack of cytosine C5 on the S-adenosyl-L-methionine (AdoMet) methyl group is concerted with formation of the Michael adduct between a conserved Cys in the active site with cytosine C6. Spontaneous and reversible proton transfer between a conserved Glu in the active site and cytosine N3 at the transition state was observed in our simulations, revealing the chemical participation of this Glu residue in the catalytic mechanism. Subsequently, the ß-elimination of the C5 proton utilizes as base an OH(-) derived from a conserved crystal water that is part of a proton wire water channel, and this syn ß-elimination reaction is the rate-limiting step. Design of novel cytosine methylation inhibitors would be advanced by our structural and thermodynamic characterization of the reaction mechanism.

Semiautomated Small-Scale Purification Method for High-Throughput Expression Analysis of Recombinant Proteins.

The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.

Aging mechanism of soman inhibited acetylcholinesterase.

Acetylcholinesterase (AChE) is a crucial enzyme in the cholinergic nervous system that hydrolyzes neurotransmitter acetylcholine (ACh) and terminates synaptic signals. The catalytic serine of AChE can be phosphonylated by soman, one of the most potent nerve agents, and subsequently undergo an aging reaction. This phosphonylation and aging process leads to irreversible AChE inhibition, results in accumulation of excess ACh at the synaptic clefts, and causes neuromuscular paralysis. By employing Born-Oppenheimer ab initio QM/MM molecular dynamics simulations with umbrella sampling, a state-of-the-art approach to simulate enzyme reactions, we have characterized the aging mechanism of soman phosphonylated AChE and determined its free energy profile. This aging reaction starts with the scission of the O2-Cα bond, which is followed by methyl migration, and results in a tertiary carbenium intermediate. At the transition state, the scissile O2-Cα bond is already cleaved with an average O-C distance of 3.2 ± 0.3 Å and the migrating methyl group is shared between Cα and Cß carbons with C-C distances of 1.9 ± 0.1 and 1.8 ± 0.1 Å, respectively. The negatively charged phosphonate group is stabilized by a salt bridge with the imidazole ring of the catalytic histidine. A major product of aging, 2,3-dimethyl-2-butanol can be formed swiftly by the reaction of a water molecule. Our characterized mechanism and simulation results provide new detailed insights into this important biochemical process.