PRE-STEADY-STATE KINETIC-ANALYSIS OF SEQUENCE-DEPENDENT NUCLEOTIDE EXCISION BY THE 3'-EXONUCLEASE ACTIVITY OF BACTERIOPHAGE-T4 DNA-POLYMERASE
PRE-STEADY-STATE KINETIC-ANALYSIS OF SEQUENCE-DEPENDENT NUCLEOTIDE EXCISION BY THE 3'-EXONUCLEASE ACTIVITY OF BACTERIOPHAGE-T4 DNA-POLYMERASE
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DOI：
10.1021/bi00190a010
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发表时间：
1994-06-21
期刊：
影响因子：
2.9
通讯作者：
BEECHEM, JM
中科院分区：
文献类型：
--
作者：
BLOOM, LB;OTTO, MR;BEECHEM, JM
通过测定荧光核苷酸类似物2-氨基嘌呤脱氧核苷单磷酸(DAPMP)从已定义序列的引物/模板中去除的动力学，考察了局部DNA序列对野生型T4DNA聚合酶校对效率的影响。测定了(1)与5‘-相邻碱基的相互作用，(2)碱基对的稳定性，(3)周围序列的G.C含量对dAPMP切除的稳态前动力学的影响。从与模板T相对的3‘-末端(AP.T碱基对)切割dAPMP的速率增加了3倍以上，其中5’-邻近AP的顺序为C<；G<；T<；A；A。从位于相同周围序列中的AP.X碱基对去除dAPMP的速率随着AP.T<；AP.A<；AP.C<；AP.G的增加而增加，这与通过T-m测量预测的这些碱基对的稳定性的降低相关。一个关键的发现是，当与C相反的错配位于4个G.C碱基对旁边时，AP的切割速度比与T相反的4个A.T碱基对的正确配对时更慢，这表明核酸外切酶错配去除的特异性可能在更大程度上是由于局部引物末端的不稳定性，而不是通过对错误碱基对的特异性识别。在聚合酶启动的反应中，在大多数但不是所有的序列背景下观察到了AP切除的两相反应动力学。快速时相的频率(30-40个S(-1))对序列上下文相对不敏感。快相速率反映了正确和错误碱基对下从熔融的引物末端切割dAPMP的速率常数，并与从单链dna中去除dAPMP的速率(65-80 S(-1))大致相当。慢相(3-13 S(-1))的速率取决于序列背景；慢相可能反映了从聚合酶到核酸外切酶活性部位的转换速度，或者可能反映了核酸外切酶活性部位中引物/模板末端从退火态到熔融态的转换。使用野生型T4DNA聚合酶和两个核酸外切酶缺陷的T4聚合酶的这些数据支持这样一个模型，在该模型中，对于错配和正确匹配的引物末端，在融化的引物3‘-末端上发生外切酶，并且其中有利于去除末端错配的碱基对的特异性取决于错配对的融化的引物3’-末端的比例比正确对的大得多。
The effects of local DNA sequence on the proofreading efficiency of wild-type T4 DNA polymerase were examined by measuring the kinetics of removal of the fluorescent nucleotide analog 2-aminopurine deoxynucleoside monophosphate (dAPMP) from primer/templates of defined sequences. The effects of (1) interactions with the 5'-neighboring bases, (2) base pair stability, and (3) G.C content of the surrounding sequences on the pre-steady-state kinetics of dAPMP excision were measured. Rates of excision dAPMP from a primer 3'-terminus located opposite a template T (AP.T base pair) increased, over a 3-fold range, with the 5'-neighbor to AP in the order C < G < T < A. Rates of removal of dAPMP from AP.X base pairs located in the same surrounding Sequence increased as AP.T < AP.A < AP.C < AP.G, which correlates with the decrease in the stabilities of these base pairs predicted by T-m measurements. A key finding was that AP was excised at a slower rate when mispaired opposite C located next to four G.C base pairs than when correctly paired opposite T next to four A.T base pairs, suggesting that exonuclease mismatch removal specificities may be enhanced to a much greater extent by instabilities of local primer termini than by specific recognition of incorrect base pairs. In polymerase-initiated reactions, biphasic reaction kinetics were observed for the excision of AP within most but not all sequence contexts. Rates of the rapid phases (30-40 s(-1)) were relatively insensitive to sequence context. Rapid-phase rates reflect the rate constants for exonucleolytic excision of dAPMP from melted primer termini for both correct and incorrect base pairs and were roughly comparable to rates of removal of dAPMP from single-stranded DNA (65-80 s(-1)). Rates of the slow phases (3-13 s(-1)) were dependent on sequence context; the slow phase may reflect the rate of switching from the polymerase to the exonuclease active site, or perhaps the conversion of a primer/template terminus from an annealed to a melted state in the exonuclease active site. These data, using wild-type T4 DNA polymerase and two exonuclease-deficient T4 polymerases, support a model in which exonuclease excision occurs on melted primer 3'-termini for both mismatched and correctly matched primer termini, and where specificity favoring removal of terminally mismatched base pairs is determined by the much larger fraction of melted-out primer 3'-termini for mispairs compared to that for correct pairs.