The Study Of Function Of DNA Modifications,and The Development Of Gene Editing Tools In Two Lower Species

DNA modifications play critical roles in the regulation of gene expression,genome architecture and stability.In the past decade,several 5-methylcytosine(5m C)-derived DNA modifications were discovered,including 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC),5-carboxylcytosine(5caC)and 5-glycerylmethylcytosine(5gm C).However,the function of these novel modifications is incompletely understood.To address the problem,we sought to study the function of DNA modifications in mammals,Chlamydomonas and Naegleria.First,we use differnet TDG mutants to discriminate and study the function of 5fC and 5caC.We found that mouse TDG N168 D and N202A(human TDG N157 D and N191A)could discriminate 5fC from 5caC in vitro,while a compromised discrimination in cells.We further interpreted the essential role of R275 of human TDG in recognition of geometry alterations of DNA minor groove,which are induced by 5fC and 5caC modifications,by penetrating into DNA duplex and contacting with modified bases directly.Next,we studied the properties of DNA glycosylases homologues ofChlamydomonas.Although no 5gm C glycosylase activity was detected,we surprisingly found 5hmC,5fC and 5caC glycosylase activities in the nuclear extract,and further identified Cr ROS1 responsible for the 5fC and 5caC activities in vitro.Based on these data,we put forward a model for DNA demethylation in Chlamydomonas.To further study the functions of DNA modifications-related proteins,including glycosylases and DNA cytosine and adenine methyltransferases,in Chlamydomonas reinhardtii,we established a Cas9 RNP-based co-selection strategy to inactivate corresponding genes.Highly active Cas9 protein was purified wiith improved yield.By inactivating gene of interest and MAA7,a selection marker gene,simutaneously,we obtained desired mutants by selecting maa7 mutant with various efficiency.Moreover,we found that the gene editing efficiency was positively correlated with its expression level,suggesting the editing efficiency was affected by the local three-dimension chromosome structure or two-dimension DNA structure.Finally,we also pursued to study the function of TET(Ten-eleven translocation)enzymes and potential corresponding DNA modifications in Naegleria gruberi.5m C and 6m A were identified in N.gruberi genome by Mass Spectrometry,while no any5 m C-derived modifications were found.We also tried to establish the gene editing tool in this species by delivering Cas9 RNP via microinjection,liposome or electroporation,while no mutant was identified or isolated.We need to further test ifCas9 works in Naegleria cells with newly developed microhomolog-mediated precise integration in Chlamydomonas.In this dissertation,we identified different enzymes involved in different DNA modifications,and gained more understanding of their properities.We further established gene editing tool in C.reinhardtii,paving the way for studying its basic biology and industrial engineering as well,and also inspiring us for further development of similar tools in Naegleria gruberi,although the initial attempt failed.