Hile Suzanne E, Eckert Kristin A
Department of Pathology, Gittlen Cancer Research Institute, The Pennsylvania State University College of Medicine, Hershey Medical Center, 500 University Drive, Hershey, PA 17033, USA.
J Mol Biol. 2004 Jan 16;335(3):745-59. doi: 10.1016/j.jmb.2003.10.075.
Microsatellite DNA sequences are ubiquitous in the human genome, and mutation rates of these repetitive sequences vary with respect to DNA sequence as well as length. We have analyzed polymerase-DNA interactions as a function of microsatellite sequence, using polypyrimidine/polypurine di- and tetranucleotide alleles representative of those found in the human genome. Using an in vitro primer extension assay and the mammalian DNA polymerase alpha-primase complex, we have observed a polymerase termination profile for each microsatellite that is unique to that allele. Interestingly, a periodic termination profile with an interval size (9-11 nucleotides) unrelated to microsatellite unit length was observed for the TC and TTCC templates. In contrast, a unit-punctuated polymerase termination profile was found for the longer polypurine templates. We detected strong polymerase pauses within the TC allele at low reaction pH which were eliminated by the addition of deaza-dGTP, consistent with these specific pauses being a consequence of triplex DNA formation during DNA synthesis. Quantitatively, a strand bias was observed in the primer extension assay, in that polymerase synthesis termination is more intense when the polypurine sequence serves as the template, relative to its complementary polypyrimidine sequence. The HSV-tk forward mutation assay was utilized to determine the corresponding polymerase alpha-primase error frequencies and specificities at the microsatellite alleles. A higher microsatellite polymerase error frequency (50x10(-4) to 60x10(-4)) was measured when polypurine sequences serve as templates for DNA synthesis, relative to the polypyrimidine template (18x10(-4)). Thus, a positive correlation exists between polymerase alpha-primase pausing and mutagenesis within microsatellite DNA alleles.
微卫星DNA序列在人类基因组中普遍存在，这些重复序列的突变率因DNA序列以及长度而异。我们使用代表人类基因组中发现的多嘧啶/多嘌呤二核苷酸和四核苷酸等位基因，分析了作为微卫星序列函数的聚合酶-DNA相互作用。使用体外引物延伸测定法和哺乳动物DNA聚合酶α-引发酶复合物，我们观察到每个微卫星的聚合酶终止图谱对于该等位基因是独特的。有趣的是，对于TC和TTCC模板，观察到具有与微卫星单位长度无关的间隔大小（9-11个核苷酸）的周期性终止图谱。相比之下，对于较长的多嘌呤模板，发现了单位间断的聚合酶终止图谱。我们在低反应pH值下在TC等位基因内检测到强烈的聚合酶停顿，通过添加脱氮-dGTP消除了这些停顿，这与这些特定停顿是DNA合成过程中三链DNA形成的结果一致。定量地，在引物延伸测定中观察到链偏向，即当多嘌呤序列作为模板时，相对于其互补的多嘧啶序列，聚合酶合成终止更强烈。利用HSV-tk正向突变测定法来确定微卫星等位基因处相应的聚合酶α-引发酶错误频率和特异性。当多嘌呤序列作为DNA合成的模板时，相对于多嘧啶模板（18×10(-4)），测量到更高的微卫星聚合酶错误频率（50×10(-4)至60×10(-4)）。因此,聚合酶α-引发酶停顿与微卫星DNA等位基因内的诱变之间存在正相关。
