ABS 059: Investigating the regulation of the DNA repair protein PARP-1 by the nuclear kinesin KIF4A

Sarah Adams ¹ ² , Anju Yadav ² ³ , Radhika Subramanian ² ³

¹ Northeastern University, Department of Bioengineering
² Massachusetts General Hospital, Department of Molecular Biology
³ Harvard Medical School, Department of Genetics.

Van Wickle (2025) Volume 1, ABS 059

Introduction: Kinesin family member 4A (KIF4a) is a multi-functional motor protein involved in both chromosome and microtubule organization during cell division. In contrast to most kinesins, KIF4a localizes to the nucleus during interphase, but the nuclear functions of this protein are not well known. Prior work suggests that KIF4a interacts with and regulates the activity of poly (ADP-ribose) polymerase (PARP), a well characterized DNA repair enzyme that plays additional roles in transcription regulation and cell homeostasis. It has been hypothesized that KIF4a binding maintains PARP1 in an inactive state, and that downstream phosphorylation may disrupt this binding, thereby activating PARP1 to carry out its DNA repair functions. A mutation in KIF4a that is associated with abnormal neurological phenotypes including epilepsy, is proposed to act by altering the KIF4a-PARP1 binding a nity. However, the mechanisms by which KIF4a binds PARP1 and regulates its nuclear activities is unknown. To address this knowledge gap, I am working to reconstitute the interaction between KIF4a, and PARP-1 using puri ed proteins and de ne the interacting domains between the proteins. Next, I will examine how KIF4a modulates the DNA binding and enzymatic activities of PARP-1. Finally, I will decipher how patient mutations in KIF4a impact PARP-1 binding and regulation. Collectively, our findings will uncover a poorly understood aspect of KIF4a’s nuclear function as a PARP-1 regulator and how these functions are altered by patient mutations linked to aberrant neuronal morphology and seizure susceptibility. 

Methods: The reconstitution strategy includes heterologous expression and affinity purification of the motor protein KIF4a and the DNA repair enzyme PARP1. Protein identity and integrity are confirmed via mass photometry and DNA-binding assays. Functional characterization of each protein, as well as their interactions with DNA substrates, will be carried out using atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF) microscopy. To investigate potential protein-protein interactions and their regulation under different biochemical conditions, pulldown assays will be performed.