Construction and functional analysis of luciferase reporter plasmids containing ATM and ATR gene promoters

Abstract

In eukaryotic cells, maintenance of genomic stability relies on the coordinated action of a network of cellular processes, including DNA replication, DNA repair, cell-cycle progression, and others. The DNA damage response (DDR) signaling pathway mediated by the ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) kinases is the central regulator of this network in response to DNA damage. The serine/threonine kinases ATM and ATR are the main kinases activated following various assaults on DNA. In this study, human ATM and ATR promoter luciferase reporter constructs were generated by PCR amplification. Then both PCR fragments respectively were digested and cloned into pGL3 vector. Finally, these promoter sequences were verified by sequencing. These results showed that luciferase reporter with ATM and ATR promoters were successfully constructed. Then the activation of the ATM promoter and ATR promoter following UV light treatments were detected in A431 cells by luciferase reporter assays. The results showed that UV damage could enhance transcriptional activity of ATM/ATR. Our research will provide useful tools for further deciphering ATM/ ATR signaling and the pathways mediating the DNA damage response.