Single-molecule microscopy reveals new insights into nucleotide selection by DNA polymerase I

Abstract

The mechanism by which DNA polymerases achieve their extraordinary accuracy has been intensely studied because of the linkage between this process and mutagenesis and carcinogenesis. Here, we have used single-molecule fluorescence microscopy to study the process of nucleotide selection and exonuclease action. Our results show that the binding of Escherichia coli DNA polymerase I (Klenow fragment) to a primer-template is stabilized by the presence of the next correct dNTP, even in the presence of a large excess of the other dNTPs and rNTPs. These results are consistent with a model where nucleotide selection occurs in the open complex prior to the formation of a closed ternary complex. Our assay can also distinguish between primer binding to the polymerase or exonuclease domain and, contrary to ensemble-averaged studies, we find that stable exonuclease binding only occurs with a mismatched primer terminus.

Single-molecule DNA repair in live bacteria

“…The total Pol repair time includes filling the single-nucleotide gap and potential strand displacement synthesis at a rate of >10 nt/s (36), followed by nuclease cleavage of the generated 5′ flap and product release; the latter is the likely single rate-limiting step. Our in vivo result is in agreement with the ∼2-s binding time of Klenow fragment (large domain of Pol) to a DNA primer in vitro (37). The Lig repair time corresponds to the ligation reaction and product release and is consistent with the in vitro measurement of 2.4 s (38).…”

Single-molecule Investigation of Substrate Binding Kinetics and Protein Conformational Dynamics of a B-family Replicative DNA Polymerase

“…However, it is apparent that binding to the polymerase site still accounts for a significant portion of binding events with the triple mismatch at the primer-template junction. This result is in contrast to those from similar studies that suggest that KF binds DNA exclusively in the exonuclease site in the presence of a double mismatch (29). This difference between KF and PolB1 in the preference of mismatched DNA for binding to the exonuclease site may be the result of structural differences between the enzymes including the interactions between the finger and exonuclease domains that are unique to crenarchaeal B-family DNA polymerases such as PolB1 (36).…”

“…Additionally, as with the other archaeal B-family DNA polymerases (37)(38)(39)(40), the exonuclease domain of PolB1 lies on the opposite side of the palm domain than observed for DNA polymerases from other families. Furthermore, the three mutations required to inactivate the exonuclease of PolB1 (see "Experimental Procedures") may be more disruptive to DNA binding to the exonuclease site than the single mutation used to remove this activity in KF (29). Nevertheless, our studies clearly demonstrate that binding to the exonuclease site of PolB1 is increasingly favored with increasing destabilization of the primer-template terminus.…”

“…4B). To determine the effect of the concentration of PolB1 on the DNA binding kinetics, we made use of a technique known as PIFE in which the binding of an unlabeled DNA polymerase to Cy3-labeled DNA has been shown to lead to an increase in fluorescence intensity (26,29). This technique allows for the use of protein concentrations above what can be used for single-molecule FRET as we have observed that concentrations above ϳ15 nM Cy5-labeled PolB1 result in a high background fluorescence that makes data analysis difficult.…”

“…Interestingly, single-molecule total internal reflection fluorescence studies have also yielded conflicting results about whether or not KF is able to bind fully complementary DNA in an editing mode with the primer bound to the exonuclease active site. One study reported that the binding to the exonuclease site was observed in ϳ13% of all binding events to fully matched DNA (30), whereas another suggested that binding to the exonuclease site was only possible in the presence of a mismatched primer terminus (29). Although single-molecule methods have begun to illuminate mechanistic aspects of the activities of several DNA polymerases that cannot be easily investigated by ensemble measurements, further study is necessary to resolve aforementioned discrepancies and to provide additional mechanistic insights.…”