Structural and Functional Studies of Drp1, a Rint1 Family Protein in Fission Yeast Schizosaccharomyces pombe

Abstract

Cell is the basic building block of all living organism, broadly divided into prokaryotic cell and eukaryotic cells. Robert Hook a Scottish botanist was known to discover “cell” in 1665 and also recognize the nucleus first time. The botanist Matthias Jakob Schleiden (1838) and zoologist Theodor Schwann (1839) proposed his famous hypothesis “cell theory” and suggest that the cells are the structural element of plants and animals (Mazzarello, 1999). Cells have property that they can divide into daughter cells by a highly regulated and complex process known as mitotic cell cycle. For this, the cells double their genomic content by DNA Replication. During this process the DNA of the cell is at higher risk of getting altered by various endogenous chemical agents generated during cellular metabolism such as free radicals, reactive oxygen species (ROS) and other metabolites as well as exogenous chemical and ionizing radiation (Mazouzi et al., 2014). Alteration in DNA structure either due to strand break, missing of base or chemically changed base causes DNA damage (Charames and Bapat, 2003). To maintain the normal cellular process and genomic stability, cells have surveillance mechanism known as cell cycle checkpoints. Checkpoint is a highly controlled mechanism that ensures the fidelity of cell division in eukaryotic cells. There are three major cell cycle checkpoints; G1/S checkpoints, intra S-checkpoints and at the G2/M checkpoints (Barnum and O'Connell, 2014). The genetic and epigenetic changes such as deletion, mutation, duplication, insertion, and chromosome aberrations and translocation may affect the regulation and function of gene product that leads to loss and gain of function. These changes are the major cause of cancer (Baylin et al., 2001). DNA double strand breaks (DSBs) are the most harmful form of DNA lesion, arise due to various reasons including replication fork collapse. If the DNA breaks remain unrepaired they can also leads to chromosomal aberrations, genomic instability and cancer. In order to repair DNA double strand break, the cells halts the cell cycle progression and generate signal to repair damaged DNA (Harrison and Haber, 2006). The Double strand break is either repaired by homologous recombination (HR) or Non-Homologous end joining (NHEJ) (Davis and Chen, 2013; Sung and Klein, 2006). Non homologous end joining (NHEJ) is an error– prone repair pathway mainly predominates in G0 and G1 phase of cell cycle. The proteins involved in this repair pathway are DNA dependent protein kinase (DNAPK), serine /threonine protein kinase, XRCC4, Ku and DNA ligase IV that promotes direct ligation of DSBs (Lees-Miller and Meek, 2003). Homologous recombination (HR) is regulated by the protein belongs to RAD52 epistasis group such as RAD51, RAD52, RAD54, RAD59 and MRE11, RAD50, NBS1/XRS2 (MRN/X) complex and repair the damage site using the homologous sequence (Li and Heyer, 2008). MRN/X complex is a multifunctional protein complex involved in many pathways such as Spo11 mediated programmed double strand breaks generation during meiosis for proper chromosome segregation, DNA damage repair, activation of checkpoints; telomere maintenance and DNA replication (Gobbini et al., 2016; Milman et al., 2009). It acts as a sensor of double strand break and bind to the damage site to activate the downstream signaling pathway such as ataxia telangiectasia mutated protein (ATM), the ATM and Rad3-related kinase (ATR) phosphorylate the effector molecule Chk2 and Chk1 respectively (Lamarche et al., 2010). Rad50 is an important component of MRN/X complex, highly conserved among species and belongs to the structural maintenance of chromosome protein family. More than 50% sequence is identical in human and yeast Rad50 (Hopfner et al., 2000). N and C-terminus domain of Rad50 contains ATPase motif, which is connected by a long coiled coil structure. The centre of the coiled coil structure contains a conserved CXXC residue (Ccysteine, X- hydrophobic amino acids), involved in Zn2+ dependent dimerization of Rad50 (Rojowska et al., 2014).