Borek Arkadiusz, Bonarek Piotr, Kowalska Ewa, Banaś Agnieszka Katarzyna, Strzałka Wojciech
Department of Plant Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
Department of Physical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
BMC Plant Biol. 2025 May 16;25(1):648. doi: 10.1186/s12870-025-06671-y.
Flap endonuclease 1 (FEN1) is part of a group of nuclear enzymes involved in eukaryotic DNA replication and repair. In our studies, using both biochemical and biophysical approaches, we demonstrated that Arabidopsis thaliana FEN1 (AtFEN1) is unstable and prone to aggregation. To understand the reasons for AtFEN1 aggregation, we first analyzed the effects of heparin sodium and sodium chloride on its aggregation. We found that both heparin sodium and sodium chloride modulated the aggregation of this enzyme; however, achieving the same level of aggregation inhibition required using a sodium chloride concentration five orders of magnitude higher than that of heparin. Subsequently, to identify potential nuclear factors that may modulate the biological activity of AtFEN1 in vivo, we used DNA. Our experiments showed that negatively charged double-stranded DNA (dsDNA), similarly to the double-flap DNA (dfDNA) substrate of AtFEN1, inhibited AtFEN1 aggregation. This inhibitory effect was much less pronounced when single-stranded DNA (ssDNA) was used. Moreover, dfDNA prevented the loss of biological activity of AtFEN1. Finally, we revealed that AtFEN1 aggregation was also blocked by Arabidopsis proliferating cell nuclear antigen 1 (PCNA1), a natural interacting protein of AtFEN1. However, this effect was observed only when the putative PCNA-interacting protein (PIP)-box sequence was present in AtFEN1.
瓣内切核酸酶1（FEN1）是参与真核生物DNA复制和修复的一组核酶的一部分。在我们的研究中，使用生化和生物物理方法，我们证明了拟南芥FEN1（AtFEN1）不稳定且易于聚集。为了了解AtFEN1聚集的原因，我们首先分析了肝素钠和氯化钠对其聚集的影响。我们发现肝素钠和氯化钠都调节了这种酶的聚集；然而，要达到相同水平的聚集抑制，所需的氯化钠浓度比肝素高五个数量级。随后，为了鉴定可能在体内调节AtFEN1生物活性的潜在核因子，我们使用了DNA。我们的实验表明，带负电荷的双链DNA（dsDNA）与AtFEN1的双瓣DNA（dfDNA）底物类似，抑制了AtFEN1的聚集。当使用单链DNA（ssDNA）时，这种抑制作用不太明显。此外，dfDNA阻止了AtFEN1生物活性的丧失。最后，我们发现AtFEN1的聚集也被拟南芥增殖细胞核抗原1（PCNA1）阻断，PCNA1是AtFEN1的天然相互作用蛋白。然而，只有当AtFEN1中存在假定的PCNA相互作用蛋白（PIP）框序列时，才会观察到这种效果。
