Biology subjects

Hussen, A.

Publications and source records attributed to Hussen, A..

2 recordsLinked to original sources

bioRxiv2023

The VEGF G-quadruplex forming promoter is repaired via long-patch BER

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased [~]2-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol {beta} performs multiple insertions on G4 substates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol {beta} products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol {beta} prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.

biochemistry↗

bioRxiv2021

Effects of α-crystallin gene knockout on zebrafish lens development

The -crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the -crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with null mutations for each of the three -crystallin genes (cryaa, cryaba and cryabb). The absence of normal protein was confirmed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of A-crystallin produced a variety of lens defects with varying severity in larval lenses at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of either Ba- or full-length Bb-crystallin produced no substantial lens defects. Mutation of each -crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for A-crystallin in lens development, but the range of phenotype severity suggests that its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.

developmental biology↗