Genomic stability and DNA repair are key factors in human health. When repair mechanisms are defective, humans become predisposed to a range of cancers. One kind of DNA damage for which effective repair is vital to humans, crosslinking of DNA. This occurs when various agents react with two different position in the DNA; in opposite strands of the DNA it is known as an interstrand crosslink (ICL). The presence of ICL results in replication arrest and cell death if the crosslink is not repaired. Repair of the ICL is highly complicated and involves a number of factors. Defects in repair mechanisms are seen in human disorders such as Fanconi anaemia and Werner syndrome. Our investigation focuses on the role of two related 3’-5’ endonucleases, EXDL2 and WRN exonuclease. Mainly focusing on the exact mechanism of expression in certain stages of the cell cycles in flies which are double mutant for both exonucleases. Hence providing a deeper knowledge of ICL repair process relating to ageing and cancer therapy.
Interstrand crosslinks (ICLs) are induced by different chemical agents which result in complementary strands of DNA becoming covalently bonded. These highly toxic DNA lesions negatively affect a series of normal DNA copying processes; such as transcription and replication by inhibiting DNA strand separation. Hence, leading to cell death.
This project exploits the genetic similarities between the fruit fly (Drosophila melanogaster) and humans in order to investigate and understand the role of the two related 3’-5’ exonucleases, CG7670 and CG6744 exonuclease, in the repair of interstrand crosslinks (ICL) in the fruit fly. Based on protein sequence and biochemical similarity the two exonucleases are closely associated with it human counterpart hence have been designated as DMWRNexo and DmEXDL2 respectively.
We can make use of genetic markers to reveal genomic instability in flies, DNA damage can be confirmed by the presence of physical characteristics (phenotype). Normal hair phenotype shows a single hair per cell on the wing surface, abnormal phenotype is represented by seeing two or more wing hairs per cells Fig 1.
It has been previously established that flies mutant for EXDL2 (DmEXDL2) have elevated rates of genomic instability resulting from chromosome breakage and loss of the acentric fragments, in contrast to WRNexo (DmWRNexo) mutants which results in increased mitotic exchange, both exonucleases play significant roles in the repair of ICL.
Further assessment of the double mutant will help to determine if the two exonuclease operate on the same or different pathway of DNA repair in general and ICL repair in particular. Their similarities will be explored specifically the presence of expression in certain moments of the cell cycle. Cell cycle specificity can be investigated by analysing genetic and biochemistry interaction with known protein in the standard pathway of DNA repair.
These findings will have significant implications in understanding the complex process of ICL repair in humans. This repair process to damaged DNA is well-regulated, however when the damage gets to a critical level, cell division halts preventing continuous replication of damage cells which is seen in normal ageing. Moreover, in Werner syndrome, a disorder that results in premature ageing (adult progeria), patients have a defect (mutation) in the WRN gene (see fig2).
Werner syndrome is characterized by the appearance of premature aging, therefore used as a model for understanding normal ageing process. In addition research into ICL repair mechanism enhances the understanding of cancer therapies that rely on ICL induction, such as genotoxic chemotherapy where ICL induction is used to kill highly proliferative cancer cells and could provide novel insights into drug resistance. Alternatively WRN and EXDL2 inhibitor can be explored as a possible therapeutic agents, resulting in a combination therapy which requires very low doses of extremely toxic crosslinking agents, hence might have great potentials for developing safer and more effective strategies for cancer.