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...
Seed size critically affects grain yield of crops and hence represents a key breeding target. The development of embryo-nourishing endosperm is a key driver of seed expansion. We here report unexpected dual roles of the transcription factor EIN3 in regulating seed size. These EIN3 functions have remained largely undiscovered because they oppose each other. Capitalizing on the analysis of multiple ethylene biosynthesis mutants, we demonstrate that EIN3 represses endosperm and seed development in a pathway regulated by ethylene. We, in addition, provide evidence that EIN3-mediated synergid nucleus disintegration promotes endosperm expansion. Interestingly, synergid nucleus disintegration is not affected in various ethylene biosynthesis mutants, suggesting that this promoting function of EIN3 is independent of ethylene. Whereas the growth-inhibitory ethylene-dependent EIN3 action appears to be encoded by sporophytic tissue, the growth-promoting role of EIN3 is induced by fertilization, revealing a generation conflict that converges toward the key signaling component EIN3.
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