Localization Of Cytosine Modification In DNA By Deaminase-mediated Analysis

The completion of the Human Genome Project marks the increasing importance of genome sequencing research in the field of life sciences.5-methylcytosine(5m C)is the most common type of DNA methylation and accounts for approximately 3-8%of total cytosine in mammals.Under the oxidation of the Tet protein,5-methylcytosine is gradually oxidized to 5-hydroxymethylcytosine(5hm C),5-formylcytosine(5f C)and 5-carboxycytosine(5ca C).In order to gain insight into the biological function and significance of each cytosine methylation modification,it is of great significance to analyze its distribution on the whole genome.These modifications have important functions of epigenetic regulation.In order to further reveal the biological functions of these modifications,it is necessary to accurately clarify their exact positions on the genome and obtain their single-base resolution localization information.Apolipoprotein B mRNA editing enzyme,catalytic polypeptide-like(APOBEC)family proteins have deamination activity of cytosine.APOBEC3 A protein can deaminate cytosine and 5m C on DNA,and also has certain deaminating activity for oxidation derivatives of 5m C.The main purpose of this thesis is to study the deamination activity of APOBEC3 A protein on C?5m C?5hm C?5f C and 5ca C.The single base resolution analysis of 5hm C and 5ca C in DNA was performed by using the deamination activity of APOBEC3 A protein.1.single-nucleotide resolution analysis of 5hm CWe first preparepd and purified the recombinant APOBEC3 A protein.The activity of APOBEC3 A protein was identified by restriction endonuclease method and high performance liquid chromatography tandem mass spectrometry analysis.APOBEC3 A can easily deaminate C,5m C and 5hm C to the corresponding uracil(U),thymine(T)and 5-hydroxymethyluracil(5hm U).In order to detect 5hm C on DNA,we used the T4 bacteriophage ?-glucosyltransferase to label 5hm C on DNA.The labeled 5hm C can resist the deamination of APOBEC3 A.After PCR amplification,C and 5m C on the original DNA template were read as T,and the protected 5hm C was read as C during the sequencing process.Combined with second-generation sequencing,the single base localization information of 5hm C on genomic DNA can be obtained.2.single-nucleotide resolution analysis of 5ca CThe reaction rate of APOBEC3 A protein to C,5m C,5hm C,5f C and 5ca C was investigated by enzymatic reaction kinetics.We found that APOBEC3 A has almost no deamination activity on 5ca C.Based on the results,we directly used APOBEC3 A to treat DNA.In this respect,C,5m C,5hm C and 5f C were deaminated by APOBEC3 A protein to form the corresponding deamination product.After PCR amplification,C,5m C,5hm C and 5f C were read as T in the sequencing process,only 5ca C was read as C.By enriching 5ca C in genomic DNA,combined with second-generation sequencing,single-base localization information of 5ca C can be obtained.In summary,we established the single base resultion methods by employing APOBEC3 A deaminase in conjugation with sequencing for the detection of 5hm C and5 ca C in DNA,which may facilite the functional studies of 5hmC and 5caC.