Selectivity of DNA Replication

Andreas Marx, Daniel Summerer, and Michael Strerath 

All DNA synthesis required for DNA repair, recombination, and replication depends on the ability of DNA polymerases to recognize the template and correctly insert the complementary nucleotide. The mechanisms whereby these enzymes achieve this tremendous task has been a central topic of interest since the discovery of the first DNA polymerase, E. coli DNA polymerase I, by Arthur Kornberg approximately half a century ago [1], Since then enormous efforts from scientists in many disciplines have been undertaken to gain insights into the complex mechanisms and functions of these molecular machines. 

Valuable insights into how DNA polymerases process their substrates were obtained as a result of detailed kinetic studies of the enzymes. Benkovic and coworkers employed rapid quenching techniques to study the kinetics of transient intermediates in the reaction pathway of DNA polymerases [5]. Intensive studies revealed that E. coli DNA polymerase I follows an ordered sequential reaction pathway when promoting DNA synthesis. Important aspects of these results for DNA polymerase fidelity are conformational changes before and after the chemical step and the occurrence of different rate-limiting steps for insertion of canonical and non-canonical nucleotides. E. coli DNA polymerase I discriminates between canonical and non-canonical nucleotide insertion by formation of the chemical bond. Bond formation proceeds at a rate more than several thousand times slower when an incorrect dNTP is processed compared with canonical nucleotide insertion. 

Taking together, DNA polymerase structural data indicate a high degree of shape complementary between the active sites of the enzymes and the nucleotide substrates, suggesting that geometrical constraints are at least one cause of DNA polymerase fidelity. 

These assumptions are further supported by the finding that mutations that are believed to alter the geometry of the binding pocket or the conformational changes 

---

The fidelity of DNA replication is maintained by (1) base selection by the polymerase, (2) a 3 —>5 proofreading exonuclease activity that is part of most DNA polymerases, and (3) specific repair systems for mismatches left behind after replication. 

Poater, J. Swart, M. Guerra, C. R Bickelhaupt, R M. Selectivity in DNA replication. Interplay of steric shape, hydrogen bonds, Jt-stacking and solvent effects, Chem. Commun. 2011, 47, 7326-7328. 

DNA-DNA and DNA-protein cross-links inhibition of DNA replication selective cytotoxicity for myeloid cells crosses blood-brain barrier... 

DNA, a phase DNA, or an SV40 DNA, which are cleaved asymmetrically at specific sequences by a certain restriction enzyme.After joining the DNA segment to the cloning vehicle, the resulting hybrid DNA (known as a chimera) can be used to transform a suitable cell. The hybrid DNA can then be selected from among the transform cells known as clones, and its expression studied in terms of DNA replication, transcription, or translation. 

The consequence of ADA deficiency is accumulation of adenosine and 2 -deoxyadenosine, substances toxic to lymphocytes, important cells in the immune response. 2 -Deoxyadenosine is particularly toxic because its presence leads to accumulation of its nucleotide form, dATP, an essential substrate in DNA synthesis. Elevated levels of dATP actually block DNA replication and cell division by inhibiting synthesis of the other deoxynncleoside 5 -triphosphates (see Chapter 27). Accumulation of dATP also leads to selective depletion of cellular ATP, robbing cells of energy. Children with ADA SCID fail to develop normal immune responses and are susceptible to fatal infections, unless kept in protective isolation. 

---