The N-terminus region of Drp1, a Rint1 family protein is essential for cell survival and its interaction with Rad50 protein in fission yeast S.pombe
Introduction
Fission yeast Drp1 (damage responsive protein 1), a RINT1/TIP1 family protein exhibit 30% sequence similarity with human Rint1 which has been reported as a Rad50 interacting protein involved in radiation-induced G2/M checkpoint [1]. The homozygous deletion of a rint1 allele in mice results in early embryonic death due to defects in maintaining the Golgi dynamics and centrosome integrity [2]. We have also previously shown that the spores containing a deletion of drp1, S. pombe ortholog of rint1 gene are unable to germinate and might be arrested in interphase without undergoing cell division [3]. Also, Rb-related p130 protein forms a complex with Rad50 through Rint1 and regulates telomerase independent maintenance of telomere length [4]. N-terminal region of human Rint1 interacts with ZW10 protein, a dynamitin–interacting protein that binds to the kinetochore and plays a role in spindle checkpoint activation [5]. Rint1-ZW10 also interacts with the COG complex and regulates SNARE complex assembly for endosome to trans-Golgi network trafficking [6].
Defects in DNA replication or DNA repair process can result in DNA double-strand breaks which are the most harmful form of a lesion. The unrepaired DNA lesion leads to genomic instability which is the major cause of tumor development [7]. The double-strand break is either repaired by homologous recombination or non-homologous end joining. Non-homologous end joining (NHEJ) is an error-prone repair pathway that mainly predominates in the G0 and G1 phase of the cell cycle [8,9]. The proteins involved in this repair pathway are DNA dependent protein kinase (DNA-PK), a serine /threonine protein kinase, XRCC4, Ku, and DNA ligase IV that promotes direct ligation of DSBs [10]. Homologous recombination (HR) is regulated by the protein that belongs to the RAD52 epistasis group such as RAD51, RAD52, RAD54, RAD59 and MRE11, RAD50, NBS1/XRS2 [11,12]. The Rad50-Mre11-Nbs1 (MRN) complex acts as a sensor of DNA double-strand break and plays a critical role in DNA repair, checkpoint activation, and telomere maintenance [13,14]. Rad50, an important component of MRN complex displays sequence and structural similarity to the structural maintenance of chromosome (SMC) family of proteins [15]. The N and C-terminus domain of Rad50 contains the ATPase motif, which is connected by a long coiled-coil structure containing a zinc hook (CxxC) motif that involves in Zn2+ dependent dimerization of Rad50 [16,17]. Here we have studied the structural functional analysis of Drp1 and Rad50 protein and their effect on DNA damage response.
Section snippets
Yeast strain, growth conditions, and cloning
The yeast strains used during this study were constructed using standard genetic methods and listed in Table 1. For spotting assays, cells were grown at 25 °C till mid-log phase, serially diluted and spotted on plates containing MMS, for UV sensitivity the plates were irradiated with the indicated dose of UV light and incubated at 25 °C for 3–4 days before taking photographs. For temperature sensitivity assay plates were incubated at 36 °C before taking photographs. A detailed method for the
The N-terminus region of drp1 is essential for the survival
Previously, we have reported that drp1 is an essential gene and is required for recovery from DNA damage [3]. To further characterize the role of Drp1 we constructed plasmid containing truncations of drp1 in frame with C-terminus HA tag (Fig. 1A). The plasmids were transformed into a heterozygous diploid strain carrying a deletion of the chromosomal drp1 gene. After sporulation and random spore analysis, the haploid cells carrying drp1 deletion along with drp1 truncation plasmids were selected.
Discussion
Cells have a mechanism that ensures the accuracy of cellular events to maintain genomic integrity. Despite the tight regulatory mechanisms, the genomic instability can arise due to the defects in DNA damage repair and DNA replication pathway [32]. Any defect in replication fork progression can lead to changes in DNA structure generating DNA double-strand breaks [33] which are the most harmful form of DNA lesion that may result in genomic instability and cancer [34]. Fission yeast Drp1, a
Conclusions
The haploid S.pombe cells lacking the N-terminus region of Drp1 were unable to survive indicating that the N-terminus region is essential for its survival. The C-terminus truncation mutant drp1C1Δ and drp1C2Δ that contain residues from 1 to 347 and 1–580 respectively exhibit temperature-sensitive phenotype. The genetic interaction with rad50 and accumulation of Rad52-YFP foci in drp1C1Δ, drp1C2Δ truncation mutants suggest their role in the DNA damage repair pathway. We have shown that the
Author statement
SG, RR design and performed the experiments with the help of PPM.
JK and MIS have performed bioinformatics analysis.
KD and Ashish have performed SAXS analysis.
SA has conceived and designed the experiments and also wrote the manuscript with the help of Ashish and MIS.
Declaration of Competing Interest
None.
Acknowledgement
We thank the members of our lab for helpful discussions and technical support. We are grateful to Dr. Satish Mishra and Rima Ray Sarkar for the technical help provided in fluorescence microscopy work. This work was supported by the grant from the Science and Engineering Research Board (SERB), India (EMR/2016/000749) and Council of Scientific and Industrial Research, New Delhi, India. SG acknowledges the Department of Biotechnology (DBT) for providing research fellowship. The CDRI communication
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CSIR-Institute of Microbial Technology, Sector 39A Chandigarh, India.