Plevani P, Chang L M
Biochemistry. 1978 Jun 27;17(13):2530-6. doi: 10.1021/bi00606a012.
In vitro DNA synthesis by yeast DNA polymerase I can be initiated by partially purified yeast RNA polymerases in the presence or absence of rNTPs. Homogeneous yeast RNA polymerase I initiates DNA synthesis by yeast DNA polymerase I on single-stranded DNA templates only in the presence of all four rNTPs. A protein capable of initiating enzymatic DNA synthesis on single-stranded DNA in the absence of rNTPs has also been separated from partially purified yeast RNA polymerase I fractions. Analysis of the RNA polymerase I initiated replication products of phage fd DNA on alkaline sucrose gradients showed noncovalent linkage between the newly synthesized DNA and the template. Isopycnic analyses of the ribonucleotide initiated fd DNA replication products demonstrated covalent linkage between the initiator RNA and newly synthesized DNA. Results from 32P-transfer experiments confirmed the covalent linkage between RNA and DNA chains and showed the presence of all four ribo- and deoxyribonucleotides at the RNA--DNA junctions. The ribonucleotide found most frequently at the RNA--DNA junction is uridylate and the purine deoxynucleotides occur more frequently than pyrimidine deoxynucleotides.
酵母DNA聚合酶I的体外DNA合成可由部分纯化的酵母RNA聚合酶在有或无核糖核苷三磷酸（rNTPs）的情况下启动。纯的酵母RNA聚合酶I仅在所有四种rNTPs存在时，才能在单链DNA模板上由酵母DNA聚合酶I启动DNA合成。一种能够在无rNTPs时在单链DNA上启动酶促DNA合成的蛋白质也已从部分纯化的酵母RNA聚合酶I组分中分离出来。对噬菌体fd DNA在碱性蔗糖梯度上由RNA聚合酶I启动的复制产物的分析表明，新合成的DNA与模板之间存在非共价连接。对核糖核苷酸启动的fd DNA复制产物的等密度分析表明，引发剂RNA与新合成的DNA之间存在共价连接。32P转移实验的结果证实了RNA与DNA链之间的共价连接，并表明在RNA-DNA连接处存在所有四种核糖核苷酸和脱氧核糖核苷酸。在RNA-DNA连接处最常发现的核糖核苷酸是尿苷酸，嘌呤脱氧核苷酸比嘧啶脱氧核苷酸出现得更频繁。
