Bhattacharyya S, Griep M A
Department of Chemistry, Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0304, USA.
Biochemistry. 2000 Feb 1;39(4):745-52. doi: 10.1021/bi991555d.
The effect of DnaB helicase on the initiation specificity of primase was studied biochemically using a series of single-stranded DNA templates in which each nucleotide of the trinucleotide d(CTG) initiation sequence was systematically varied. DnaB helicase accelerated the rate of primer syntheisis, prevented "overlong" primers from forming and decreased the initiation specificity of primase. In the presence of DnaB helicase, all trinucleotides could serve as the primer initiation site although there was a distinct preference for d(CAG). These data may explain the high chromosomal prevalence of octanucleotides containing CTG on the leading strand and its complement CAG on the lagging strand. The specificity of DnaB helicase places it on the lagging strand template where it stimulates the initiation of Okazaki fragment synthesis. In the absence of DnaB helicase, primase preferentially primed the d(CTG) template. In the presence of DnaB helicase, the initiation preference was not only altered but also the preferred initiating nucleotide was found to be GTP rather than ATP, for both the d(CTG) and the d(CAG) templates. This suggested that the specificity of primase for the d(CTG) initiation trinucleotide was predominantly unaffected in the absence of DnaB helicase on short ssDNA templates, whereas in conjunction with DnaB helicase, the specificity was altered and this alteration has significant implications in the replication of Escherichia coli chromosome in vivo.
利用一系列单链DNA模板，对DnaB解旋酶对引发酶起始特异性的影响进行了生化研究，在这些模板中，三核苷酸d(CTG)起始序列的每个核苷酸都被系统地改变。DnaB解旋酶加快了引物合成的速率，阻止了“过长”引物的形成，并降低了引发酶的起始特异性。在DnaB解旋酶存在的情况下，所有三核苷酸都可以作为引物起始位点，尽管对d(CAG)有明显的偏好。这些数据可以解释在前导链上含有CTG的八聚体及其在滞后链上的互补序列CAG在染色体上的高发生率。DnaB解旋酶的特异性使其位于滞后链模板上，在那里它刺激冈崎片段合成的起始。在没有DnaB解旋酶的情况下，引发酶优先引发d(CTG)模板。在DnaB解旋酶存在的情况下，起始偏好不仅发生了改变，而且对于d(CTG)和d(CAG)模板，发现优先的起始核苷酸是GTP而不是ATP。这表明，在短单链DNA模板上，在没有DnaB解旋酶的情况下，引发酶对d(CTG)起始三核苷酸的特异性主要不受影响，而与DnaB解旋酶结合时，特异性发生改变，这种改变对体内大肠杆菌染色体的复制具有重要意义。
