Carver T E, Sexton D J, Benkovic S J
Department of Chemistry, 152 Davey Laboratory, Pennsylvania State University, University Park 16802, USA.
Biochemistry. 1997 Nov 25;36(47):14409-17. doi: 10.1021/bi971423p.
The mechanism of bacteriophage T4 DNA polymerase (gp43) and clamp (gp45) protein dissociation from the holoenzyme DNA complex was investigated under conditions simulating the environment encountered upon completion of an Okazaki fragment. Lagging strand DNA synthesis was approximated using a synthetic construct comprised of a doubly biotinylated, streptavidin-bound 62-mer DNA template, paired with complementary primers to generate an internal 12-base gap where the 5'-end primer contained either a 5'-OH (DNA primer) or a 5'-triphosphate (RNA primer) group. Rapid kinetic measurements revealed that upon encountering the blocking primer, the holoenzyme either dissociates from DNA (approximately 40%) or strand-displaces the blocking strand (approximately 60%). The two blocking oligonucleotides (DNA or RNA) induce a 30-50-fold increase in the rate of holoenzyme dissociation, with both polymerase and clamp proteins dissociating simultaneously. Inhibition of ATP hydrolysis by ATP-gamma-S did not have a measurable effect upon holoenzyme dissociation from DNA. The presence of gp32, the single-strand binding protein, caused a small (3-fold) increase in the rate constant for dissociation.
在模拟冈崎片段合成完成后所遇环境的条件下，研究了噬菌体T4 DNA聚合酶（gp43）和夹钳（gp45）蛋白从全酶-DNA复合物上解离的机制。使用由双生物素化、与链霉亲和素结合的62聚体DNA模板组成的合成构建体来近似滞后链DNA合成，该模板与互补引物配对以产生内部12个碱基的缺口，其中5'-端引物含有5'-OH（DNA引物）或5'-三磷酸（RNA引物）基团。快速动力学测量表明，遇到阻断引物时，全酶要么从DNA上解离（约40%），要么进行链置换阻断链（约60%）。两种阻断寡核苷酸（DNA或RNA）使全酶解离速率提高30至50倍，聚合酶和夹钳蛋白同时解离。ATP-γ-S对ATP水解的抑制对全酶从DNA上的解离没有可测量的影响。单链结合蛋白gp32的存在使解离速率常数略有增加（3倍）。
