Replication Of Epigenetically Modified N~6-methyladenine In Human Cells

The N~6 methyl group of adenine in DNA(N~6-methyladenine,6m A)is an emerging epigenetic mark that may play important roles in regulating gene expression and stimuli response.However,it remains unclear about the impact of 6m A on DNA replication in human cells.In this study,the strategy of next-generation sequencing in combination with double-strand shuttle vector technology was employed for in-vivo replication studies.This provides a new insight for elucidating the biological consequences of the epigenetically modified N~6-methyladenine during DNA replication in human cells.The main research contents of this paper are summarized as follows:(1)To explore the effect of 6m A on DNA replication in HEK293T cellsIn this work,we employed the strategy of shuttle plasmid combined with next-generation sequencing technology for assess ing how 6m A affect the efficiency and fidelity of DNA replication in human cells.First,using p TGFP-Hha10 as the original plasmids,we constructed plasmids containing site-specifically inserted 6m A,and the 6m A-containing plasmids were transfected into HEK293T cells for in vivo replication.The resulting progeny plasmids were extracted from the cells,followed by PCR amplification and next-generation sequencing analysis.The results indicated that the presence of 6m A did not inhibit DNA replication or induce apparent base substitution products in HEK293T cells.(2)To assess the potential roles of TLS polymerases Polηand Polιin thereplicative bypass of 6m A in human cellsTo identify the potential repair mechanisms of DNA 6m A,this work explored the role of TLS polymerases Polηand Polιin the presence of 6m A during DNA replication.First,HEK293T cells deficient in Polηor Polιconstructed by using the CRISPR-Cas9 technology were detected,and our results confirmed the successful deletion of Polηor Polιin HEK293T cells.Based on the strategy of shuttle plasmid in conjunction with next-generation sequencing technology,we further explored whether Polηand Polιwere involved in the replication of 6m A.The results showed that deletion of TLS polymerase Polηor Polιdid not affect the replicative efficiency and fidelity of 6m A in human cells.(3)To assess the potential function of TLS polymerases Polκand Polζin the replicative bypass of 6m A in human cellsThis work further explored whether the TLS polymerases Polκand Polζwere involved in regulating the replicative efficiency and fidelity of 6m A in human cells.First,Using DNA sequencing together with other biological methods to validate the mutation types of Polκor Polζdeleted by CRISPR/Cas9 technology,the results of sequencing showed the successful knockout of Polκand Polζ.Next,a strategy based on shuttle plasmids coupled with next-generation sequencing was employed to further explore the potential functions of Polκand Polζin the replication of 6m A.The results showed that depletion of Polκor Polζdid not affect the replicative efficiency of 6m A in human cells.Meanwhile,loss of Polκor Polζhad no impact on the replicative fidelity of 6m A.In summary,our results showed that 6m A neither affected the efficiency of DNA replication nor caused base mutations in human cells.Since the genetic information stored in DNA must be efficiently transmitted in a high-fidelity manner in order to ensure the normal biological processes,the presence of epigenetic modifications in DNA generally does not interfere with the efficiency and fidelity of DNA replication.Therefore,the finding that 6m A has no effect on DNA replication is consistent with its potential roles as an epigenetically modification in epigenetic regulation.In addition,since Polη,Polι,Polκand Polζhad negligible roles in replicative bypass6m A,there may be other TLS polymerases or DNA repair proteins responsible for bypassing or repairing 6m A in human cells.