INSERTION OF NUCLEOTIDES OPPOSITE APURINIC APYRIMIDINIC SITES IN DEOXYRIBONUCLEIC-ACID DURING INVITRO SYNTHESIS - UNIQUENESS OF ADENINE-NUCLEOTIDES
INSERTION OF NUCLEOTIDES OPPOSITE APURINIC APYRIMIDINIC SITES IN DEOXYRIBONUCLEIC-ACID DURING INVITRO SYNTHESIS - UNIQUENESS OF ADENINE-NUCLEOTIDES
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DOI：
10.1021/bi00288a026
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发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
STRAUSS, B
中科院分区：
文献类型：
--
作者：
SAGHER, D;STRAUSS, B
在大肠杆菌突变体上培养噬菌体，用尿嘧啶n -糖基化酶处理，制备了含有20-30个无尿嘧啶/无嘧啶（AP）位点/完整环状分子的M13 DNA。AP位点阻断大肠杆菌pol i催化的体外DNA合成，确定了4种酶合成终止的位置。T4 DNA聚合酶在假定的AP位点前终止一个核苷酸。DNA pol I， AMV[禽成髓细胞病病毒]逆转录酶和DNA聚合酶。根据特定的序列在AP病变之前或部位终止合成。插入AP位点对面的核苷酸的身份是通过在第一阶段的反应中使用T4 DNA聚合酶合成直至病变，然后在第二阶段确定延伸率来确定的。嘌呤比嘧啶更容易插入相对的AP位点，而dATP在促进这种延伸方面比dGTP更有效。在所有4种dNTP的混合物中，用AP DNA测定了dNTP向dNMP的DNA依赖性转化。从dATP中最容易产生dAMP。在体外系统中，与AP位点相反的腺嘌呤核苷酸的结合具有固有的特异性。就模型系统反映体内突变事件而言，数据表明去嘌呤化应该产生转化，去嘧啶化应该产生转变。
M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites/intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for 4 enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV [avian myeloblastosis virus] reverse transcriptase and DNA polymerase .alpha. terminate synthesis before or at the site of an AP lesion depending on the particular sequence. The identity of the nucleotide inserted opposite an AP site was determined by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all 4 dNTP''s by using AP DNA. The production of dAMP from dATP occurs most readily. There is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, the data suggest that depurination should produce transversions and depyrimidination should produce transitions.