Use of XXXX and @entity2340 fluorescence as probes in kinetic analyses of @entity10840 .

@entity13129 @entity1025 @entity10840 @entity42636 @entity377 @entity13130 @entity2340
Although the use of @entity13129 ( @entity13130 ) as a probe in stopped-flow analyses of @entity10840 ( @entity10840 ) had provided important mechanistic insight, the conditions used were limited by the location of @entity13130 and the use of a combination of @entity2340 (Trp) and @entity13130 fluorescence. This study examined different DNA substrates to identify several factors that can affect the observed signal in stopped-flow experiments. Both Trp and @entity13130 emissions were separately excited and monitored. It was found that both probes show a fast phase and a slow phase of fluorescence changes, but the direction and the amplitude vary greatly between the two probes and between different DNA substrates. Detailed analyses suggested that the location of @entity13130 in the template has a significant impact on the fluorescence properties of @entity13130 and that a location opposite the incoming @entity42636 , which has been used in all such studies in the past, is not optimal. In particular, the results show that placing @entity13130 one base after the templating base greatly enhances the signal intensity, which suggests a significant change in base stacking interactions at this position during @entity377 incorporation. These results allowed us to derive an improved set of conditions which were then used to reevaluate results from previous reports. It also allows greater freedom in the type of base pairs studied, since @entity13130 is not the templating base in the nascent base pair. Kinetic constants were determined for @entity42636 and catalytic @entity1025 . The results obtained from stopped-flow experiments were compared to results from chemical quench. Stopped flow of incorrect @entity42636 incorporation and the reverse reaction are also reported, which provide useful information to the mechanism of @entity10840 .
