The RecQ DNA helicase Rqh1 promotes Rad3^ATRkinase signaling in the DNA replication checkpoint pathway of fission yeast

Abstract

Rad3; Cds1; Chk1; ATR; genome integrity. 13Word counts: Abstract: 192 (limit: 200); Main Text: 7048 (limit: 8000). 14 15 16 2 ABSTRACT 17Rad3 is the orthologue of ATR and the sensor kinase of the DNA 18 replication checkpoint in Schizosaccharomyces pombe. Under replication stress, it 19 initiates checkpoint signaling at the forks necessary for maintaining genome 20 stability and cell survival. To better understand the checkpoint initiation process, 21 we have carried out a genetic screen in fission yeast by random mutation of the 22 genome looking for mutants with defects in Rad3 kinase signaling. In addition to 23 the previously reported tel2-C307Y mutant (1), this screen has identified six 24 mutations in rqh1 encoding a RecQ DNA helicase. Surprisingly, these rqh1 25 mutations except a start codon mutation are all in the helicase domain, indicating 26 that the helicase activity of Rqh1 plays an important role in the replication 27 checkpoint. In support of this notion, integration of two helicase-inactive 28 mutations or deletion of rqh1 generated a similar Rad3 signaling defect and 29 heterologous expression of human RECQ1, BLM and RECQ4 restored the Rad3 30 signaling and partially rescued a rqh1 helicase mutant. Therefore, the replication 31 checkpoint function of Rqh1 is highly conserved and mutations in the helicase 32 domain of these human enzymes may cause the checkpoint defect and contribute 33 to the cancer predisposition syndromes. 34 35 INTRODUCTION 36 Multiple cellular mechanisms ensure the accurate transmission of genetic 37 material from one generation to the next. These include proper repair of DNA 38 damage, protection of perturbed DNA replication forks, and the checkpoints that 39 3 coordinate DNA replication and repair with cell cycle progression. Chromosomes 40 are particularly vulnerable to DNA damage during replication as they are 41 decondensed and single-stranded and highly accessible to damaging agents. In 42 addition, other factors such as excessive trinucleotide repeats or insufficient 43supply of dNTPs can also perturb DNA replication. Therefore, DNA replication is 44 subject to exquisite regulations. One such regulation mechanism is the DNA 45 replication checkpoint (DRC). The DRC senses the problems and activates 46 cellular responses such as increased production of dNTPs, cell cycle delay, fork 47 stabilization, and suppression of late firing origins, which work in concert to 48 minimize the mutation rate and ensure accurate and complete duplication of the 49 genome before the cell division. Consistent with its importance in genome 50 stability, the DRC is conserved from yeasts to humans, and defects in the cell 51 signaling pathway cause a wide range of developmental and cancer predisposition 52 syndromes. Although debatable, mutations generated by errors in DNA 53 replication followed by mistakes in repair likely contribute significantly to 54 sporadic cancers [see reviews in references (2-4) ]. 56The current model envisages that a related set of sensor proteins in all 57...

Comprehensive mutational analysis of the checkpoint signaling function of Rpa1/Ssb1 in fission yeast

“…The partial checkpoint defect of ssb1-1 and ssb1-10 also suggests a redundant factor in checkpoint initiation. Similar to the two ssb1 mutants, our previous hus screening has identified several mutants that are defective more specifically in the DRC pathway (36,37,38). It would be interesting to investigate how the ssb1 mutants interact genetically with those hus mutants.…”

“…We have recently carried out a large-scale genetic screen in fission yeast by random mutation of the genome, looking for mutants that are defective in the DRC signaling pathway. This hydroxyurea (HU)-sensitive or hus screen has identified several previously uncharacterized mutants such as tel2-C307Y in the essential Tel2-Tti1-Tti2 (TTT) complex (36), a series of mutants of rqh1 of a RecQ helicase (37), and two mutants of the essential Smc5/6 complex (38). To our surprise, although this genome-wide screen has identified every single previously known DRC gene multiple times, it did not identify a single RPA mutant, which raises a concern about the checkpoint sensor function of RPA in vivo.…”