Harrington C, Perrino F W
Department of Biochemistry, Wake Forest University Medical Center, Winston-Salem, NC 27157, USA.
Nucleic Acids Res. 1995 Mar 25;23(6):1003-9. doi: 10.1093/nar/23.6.1003.
The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.
动物细胞中复制起点处新DNA链的起始需要引发酶从头合成RNA引物，随后DNA聚合酶α从RNA引物开始延伸。为了研究DNA聚合酶α-引发酶复合物（polα-引发酶）选择引物位点的特异性，构建了一个含有复制起始位点的天然DNA模板。将两个单链DNA（ssDNA）分子相互杂交，产生一个具有开放螺旋复制“泡”的双链DNA分子作为起始区域。Polα-引发酶识别开放螺旋区域，并在富含嘧啶核苷酸的四个特定位点起始RNA引发的DNA合成。在复制“泡”模板中最靠近ssDNA-dsDNA交界处的引发位点是起始的首选位点。使用包含首选引发位点序列的40个碱基的寡核苷酸模板，引发酶合成长度为9和10个核苷酸的RNA引物，序列为5'-(G)GAAGAAAGC-3'。这些研究表明，polα-引发酶选择特定的核苷酸序列用于RNA引物形成，并表明复制“泡”的开放螺旋结构引导polα-引发酶在ssDNA-dsDNA交界处附近起始RNA引物合成。
