Human DNA Polymerase β Mutations Allowing Efficient Abasic Site Bypass

Abstract

The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase ␤ when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase ␤ was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase ␤ and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase ␤ variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.The DNA of every cell in the human body gets damaged more than 50,000 times a day (1). The relation between DNA damage and repair has a significant effect on various cancers, neurological aberrations, and the process of premature aging (2). DNA polymerases are key enzymes that function in maintaining the integrity of the encoded genetic information in DNA replication, DNA repair, DNA recombination, and the bypassing of damages in DNA. Therefore they are central to the aforementioned interplay (3).The most frequent DNA damage observed under physiological conditions are abasic sites resulting from spontaneous hydrolysis of the bond that connects the sugar and the nucleobase in DNA (4). Guanine and adenine nucleobase residues are cleaved most efficiently, resulting in the abasic sugar moiety (AP; see Fig. 1A) with the loss of the genetic information stored in the nucleobase (5). Because these lesions are devoid of genetic information, they are potentially mutagenic. The bulk of this damage is removed by base excision repair pathway, which uses the sister strand to guide incorporation of the right nucleotide in place of the lesion (6, 7). DNA polymerase ␤ acts as the main gap-filling enzyme in base excision repair (8). The enzyme governs for selecting the right nucleotide complementary to the undamaged templating nucleotide (9).DNA polymerases have been grouped in seven different families named A, B, C, D, X, and Y and reverse transcriptases. The grouping depends on sequence homology and structural similarity (10). DNA polymerase ␤ b...

Learning from Directed Evolution: Thermus aquaticus DNA Polymerase Mutants with Translesion Synthesis Activity

“…Gieseking et al identified a variant of human DNA polymerase b from a library of more than 10 000 mutants (generated by error-prone PCR) that exhibited superior lesion-bypass ability than the wild-type enzyme. [48] It was found that a mutation that has a pronounced effect was the substitution of a negatively charged glutamic acid with a positively charged lysine. Furthermore, the presence of a large positively charged DNA binding surface has been shown to promote DNA-lesion bypass by human DNA polymerase h, an enzyme that is able to perform translesion synthesis, as well as Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4), an intensively studied translesion polymerase.…”

Solution Structure and Conformational Dynamics of Deoxyxylonucleic Acids (dXNA): An Orthogonal Nucleic Acid Candidate

“…It was observed that all tested DNA polymerases of eukaryotic, prokaryotic, and archaic origin accept dXTTP and dXATP as building blocks in primer extension reactions, except for KlenTaq DNA polymerase ( Figure 5). In addition to several commonly used DNA polymerases, we tested DNA polymerase mutants with broadened substrate spectra that had been generated either by directed protein evolution or by site-directed mutagenesis (DNA polymerase b 5P20, [31] Therminator DNA polymerase M3, [32] KlenTaq DNA polymerase DM, [33] and Taq DNA polymerase M1 [34] ). Owing to the presence of an altered configuration of the 3'-hydroxyl group of the synthesized deoxyxylose triphosphates, which is required after incorporation for DNA strand elongation, we investigated how many of the xylose nucleotides can be incorporated in a row.…”

Generation of a Mono‐ubiquitinated PCNA Mimic by Click Chemistry

“…Primer extension studies: Pol η was purchased from EnzyMax, LLC. Pol β was expressed and purified as described 24. PCNA or PCNA–Ub (100 n M ) and DNA polymerase (pol η: 4 n M , pol β: 8 n M ) were mixed and reactions were started by adding the reaction mix containing template/primer (2 n M ), 1× reaction buffer (50 m M Tris‐HCl, 0.25 mg mL −1 BSA, 1 m M DTT), MgCl 2 (5 m M ), and dNTPs (10 μ M ) for pol η, and template/primer (2 n M ), BSA (0.1 mg mL −1 ), 1× reaction buffer (50 m M Tris‐HCl, pH 7.9, 70 m M KCl, 5 % glycerol, 1 m M DTT), MnCl 2 (2 m M ) and dNTPs (15 μ M ) for pol β.…”