The Phage T4 DNA Ligase Mediates Bacterial Chromosome DSBs Repair And Improves The Survival-coupled Mutagenesis And Screening

DNA double-strand breaks(DSBs)are one of the most lethal forms of DNA damage that is not as efficiently repaired in prokaryotes.Many mutagens may cause lethal DSBs which induces the cell death and ultimately hampers the accumulation of mutations on the genome during random mutagenesis.T4 DNA Ligase has been widely used in molecular biology applications in vitro given its ability of joining both sticky and blunt ended DNA(Rossi et al.,1997).However,detailed mechanism of T4 DNA Ligase remains elusive.Attempts to crystalize T4 DNA Ligase and solve its structure all failed(Mueser et al.,2010).This paper demonstrated for the first time that T4 DNA Ligase can repair DNA double-stranded breaks(including foreign and genomic DNA)in prokaryotic cells without homologous template.This is a repair pathway similar to a non-homologous end-link(NHEJ).From phage-derived single-component T4 DNA Ligase,to partially-prokaryotic cells with Ku-LigD,to more complete and complex multi-component NHEJ repair systems in eukaryotic cells.Our findings seem to provide some ideas and clues for the evolution of NHEJ.We studied the characteristics of T4 DNA Ligase repair of E.coli genomic double-stranded breaks.T4 DNA Ligase is 5 times more efficient in repairing DSB than Mt Ku-LigD;compared to the Mt Ku-LigD protein group,T4 DNA Ligase will have a longer deletion after repair.In addition,the binding of T4 DNA Ligase to the ends of DNA breaks showed randomness,but showed a higher repair efficiency for slightly homologous cohesive ends.The nuclease inhibitor protein Gam can significantly reduce the length of the deleted fragment after T4 DNA Ligase repair DSB(the length of deletion ?3.5kb is about 72%).This was significantly different from the expression of T4 DNA Ligase alone(12%)and was also higher than that of Mt Ku-LigD(58%).Through experiments,it was verified that T4 DNA Ligase can increase the survival rate of Escherichia coli,Lactobacillus plantarum,and Pseudomonas putida under ARTP ionizing radiation,and that T4 DNA Ligase can increase the resistance of Escherichia coli to radiation-like antibiotic ciprofloxacin.After expressing T4 DNA Ligase in PHB production strain E.coli XLPHB,mutants obtained by ARTP treatment showed significant changes in PHB yield.The highest PHB content was 112%of the control strain accumulation and reached 53.87 ± 1.11%w/w of dry cell weight.T4 DNA Ligase can increase the survival rate of Escherichia coli in a lethal environment such as plasma or antibiotics,so that E.coli has the opportunity to accumulate mutations,and then evolved into mutants resistant to this lethal condition We described this mutation coupled with survival as the T4 mediated survival-coupled mutagenesis(T4SM)method.When DSBs-inducing mutagen is used to mutagenize the microorganisms for the desired purpose,T4 DNA Ligase can be used to express in the host to repair the DSBs caused by the mutagen.Hosts that repair chromosome DSBs with T4 DNA Ligase may live longer and accumulate more mutations during mutagen treatment.This will result in a larger surviving pool in one strain that can have more mutations and be screened during mutagenesis.