The Function Of Nhp10 In Saccharomyces Cerevisiae Under DNA Replication Stress And Its Mechanism

Eukaryotes transmit genetic information through DNA,DNA often encounter injury which induced by internal and external factors,resulting in gene mutation and chromosome aberration,which will lead to a variety of diseases include cancer.In order to deal with the threat of DNA damage,eukaryotic cells have evolved the mechanism of DNA damage response（DNA damage response,DDR）to ensure the genetic stability.The pathways activated by cells in response to the threat of DNA damage all belong to DDR,including activation of cell cycle checkpoints,DNA double-strand break（DNA double-strand break,DSB）damage repair,DNA single-strand break damage repair,base mismatch repair,base excision repair etc.Nhp10 is directly involved in DSB damage repair by specifically interacting with DSB induced phosphorylated histone H₂A（γ-H₂AX）.Whether Nhp10 is involved in other DDR pathways besides DSB damage repair remains unclear.In order to explore the unknown function of Nhp10,first construct nhp10△（in the case of Nhp10,Nhp10 represents the Nhp10 protein;Nhp10 represents the Nhp10gene;nhp10△represents the mutant cell of Nhp10 has been deleted）by lithium acetate transformation method,observe the growth activity of nhp10△on different DNA damage agents found that nhp10△sensitive to hydroxyurea（HU）which could induce DNA replication stress,insensitive to other types damage agents.HU makes d NTP with low level by inhibiting RNR activity,delete RNR inhibitors Sml1 and Crt1 can could increase the level of d NTP,but delete Sml1 and Crt1 on the basis of nhp10△not restore nhp10△growth in HU plate.This indicated that Nhp10 was not involved in the d NTP level pathway regulated by Sml1 and Crt1.In order to explore the mechanism of Nhp10response to HU damage,the expression of Nhp10 protein was detected by Western Blot.The result showed that the protein expression level of Nhp10 in S phase decreased during HU damage（P<0.01）.It is speculated that Nhp10 may play an important role in S phase in response to HU damage.Furthermore,the changes of DNA content in S phase of wild type W303 and nhp10△during S phase were detected by flow cytometry.The results showed that nhp10△with slowly progression during S phase under HU treatment.Mec1 phosphorylation activates the S phase checkpoint effector Rad53 to inhibit cell cycle progression to avoid abnormal DNA involvement in cell division when cells damaged by HU.Western Blot detect differences in Rad53 phosphorylation level between nhp10△and wild type W303 cell,Western Blot result showed that Rad53phosphorylation level of nhp10△was higher than that of wild type W303,so the overactivation of Rad53 of nhp10△may be the reason for its slow progression in S phase.Rad53 is activated by the DNA damage checkpoint（DDC）pathway in which Rad9 acts as mediator protein and the DNA replication checkpoint（DRC）pathway in which Mrc1,Sgs1,and Ctf18 act as mediator protein.To explore the mechanism of Nhp10 in reducing Rad53 phosphorylation activity,the interaction between Nhp10 and DDC and DRC mediator proteins was examined by epistasis and Western Blot.The results are as follows:（1）The sensitivity of nhp10△rad9△to HU was combine with nhp10△and rad9△;the sensitivity of nhp10△mrc1△to HU was similarity to mrc1△;the sensitivity of nhp10△sgs1△to HU was lower sensitive than nhp10△and sgs1△;the sensitivity of nhp10△ctf18△to HU was combine with nhp10△and ctf18△;the sensitivity of nhp10△mrc1△sgs1△to HU was similar to mrc1△sgs1△.This suggested that Nhp10 acts on the DRC pathway,acting on the same pathway as the DRC mediator proteins Mrc1 and Sgs1 and act on independent pathway with Rad9 and Ctf18.（2）The Rad53 phosphorylation level of nhp10△mrc1△was higher than that of mrc1△;the Rad53 phosphorylation level of nhp10△sgs1△was higher than that of sgs1△;the Rad53 phosphorylation level of nhp10△mrc1△sgs1△was similar to that of mrc1△sgs1△.This suggested that Nhp10 attenuates Rad53 activity by attenuated the checkpoint signal which transmitted by Mrc1 and Sgs1.To explore whether Nhp10 has a function to promote cell recovery from HU damage,the morphological and DNA content of wild type W303 and nhp10△during HU damage recovery were examined by microscopy and flow cytometry.The results are as follows:（1）Cells returned to dividing normal cells after experiencing the abnormal stage of division during the recovery of HU damage,and the proportion of dividing normal cells in wild type W303 was higher than that of nhp10△（P<0.05）.（2）Wild type W303 cell cycle progression faster than that of nhp10△during HU damage recovery.To sum up,these results indicated that Nhp10 can promote cell recovery from HU damage.Rad53 dephosphorylation by its phosphatases Pph3 and Ptc2,the relationship of Nhp10 to Rad53 dephosphorylation was examined by epistasis assay and Western Blot.The experimental results are as follows:（1）The sensitivity of nhp10△pph3△and nhp10△ptc2△to HU was combine with nhp10△and pph3△and combine with nhp10△and ptc2△.（2）Rad53 phosphorylation decreased same level in wild type W303 cell and nhp10△at 15 min which recovery from HU.These results indicated that Nhp10 promotes cell recovery from HU damage in the pathway of independent with Rad53 dephosphorylation.In conclusion,this experiment demonstrated that Nhp10 can respond to replication stress induced by HU through affects the DNA replication checkpoint activity.The mechanism is that Nhp10 acts on the DNA replication checkpoint pathway,reduces Rad53 activation via attenuates the DNA replication checkpoint signal which amplify and transmit by Mrc1 and Sgs1,it makes Rad53 active in a normal range.