Hydroxyurea and inactivation of checkpoint kinase MEC1 inhibit transcription termination and pre-mRNA cleavage at polyadenylation sites in budding yeast

Abstract

The DNA damage response (DDR) is an evolutionarily conserved process essential for cell survival. The transcription changes triggered by DDR depend on the nature of DNA damage, activation of checkpoint kinases, and the stage of cell cycle. The transcription changes can be localized and affect only damaged DNA, but they can be also global and affect genes that are not damaged. While the purpose of localized transcription inhibition is to avoid transcription of damaged genes and make DNA accessible for repair, the purpose and mechanisms of global transcription inhibition of undamaged genes are less well understood. We show here that a brief cell treatment with hydroxyurea (HU) globally inhibits RNA synthesis and transcription by RNA polymerase I, II, and III (RNAPI, RNAPII, and RNAPIII). HU reduces efficiency of transcription termination and inhibits pre-mRNA cleavage at the polyadenylation (pA) sites, destabilizes mRNAs, and shortens poly(A) tails of mRNAs, indicating defects in pre-mRNA 3′ end processing. Inactivation of the checkpoint kinase Mec1p downregulates the efficiency of transcription termination and reduces the efficiency of pre-mRNAs clevage at the pA sites, suggesting the involvement of DNA damage checkpoint in transcription termination and pre-mRNA 3′ end processing.

Characteristic effect of hydroxyurea on the higher-order structure of DNA and gene expression

“…HU inactivates ribonucleotide reductase (RNR), causing a decrease in the cellular pool of deoxyribonucleoside triphosphates, which leads to the inhibition of DNA synthesis 11 – 16 . Recently, experiments using budding and fission yeasts reported that HU globally inhibited RNA synthesis and transcription by RNA polymerase 17 . Therefore, the cytotoxic and antitumor activities of HU have been attributed to enzyme-mediated effects.…”