Rapid Segmental and Subdomain Motions of DNA Polymerase β

Abstract

DNA polymerase (pol) ␤ is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between ␣-helix N and the nascent base pair. Kinetic and structural characterization of pol ␤ has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and ␣-helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of ␣-helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol ␤ were determined. The results revealed that the segmental motion of ␣-helix N was rapid (ϳ1 ns) and far more rapid than the step that limits chemistry. Binding of Mg 2؉ and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in ␣-helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within ␣-helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of ␣-helix N are discussed from the perspective of the "open" to "closed" conformational change of pol ␤ deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors. DNA polymerase (pol)1 ␤ is a 39-kDa enzyme composed of two distinct domains of 8-and 31-kDa connected by a proteasehypersensitive hinge region. The amino-terminal 8-kDa domain possesses the 5Ј-deoxyribose phosphate lyase activity required to process the 5Ј-terminus in a DNA gap during base excision repair. The polymerase active site is part of the carboxyl-terminal 31-kDa domain (for a review see Ref. 1). The domain and subdomain organization of pol ␤ is illustrated in Fig. 1, A and B. Although pol ␤ appears to have evolved separately from other families of polymerases of known structure (2), it shares many general structural and mechanistic features with other polymerases. The polymerase domain is composed of three functionally distinguishable subdomains. The polymerase catalytic subdomain coordinates two divalent metal cations that assist the nucleotidyl transferase reaction. Two additional subdomains that have primary roles in duplex DNA binding and nascent base pair (nucleoside 5Ј-triphosphate and templating nucleotide) binding border the catalytic subdomain. These subdomains will ...

A Pre-equilibrium before Nucleotide Binding Limits Fingers Subdomain Closure by Klentaq1

“…Similar conclusions were drawn from inspection of the ratelimiting kinetics of wild-type pol ␤ and a pol ␤ variant mutated at Tyr-265, a residue that does not make any contact with the DNA or the dNTP (25,26). Fluorescence studies on pol ␤ also indicated that motions in the fingers subdomain appear to be fast, but the extent to which it is not rate-limiting was not addressed (27). The conclusions that the rate-limiting conformational change actually occurs in the closed conformation would appear to also apply to the repair enzymes, such as Klentaq1 and Klenow.…”

Why nature really chose phosphate

“…Time-resolved fluorescence (Kim et al 2003) Kirby et al 2005). However, these motional effects are not likely to represent true dynamical effects, but rather merely equilibrium thermal fluctuations that follow the Boltzmann populations in different landscapes.…”

Characterization of the Dynamics of an Essential Helix in the U1A Protein by Time-Resolved Fluorescence Measurements

“…Time-resolved fluorescence anisotropy is a well-established method to measure dynamics on the picosecond to nanosecond time scales in proteins and protein-nucleic acid complexes. [45][46][47][48][49] For example, methods that are similar to the ones reported here have been used to characterize segmental motion of R-helix N in DNA polymerase . 47 We performed experiments with both Lys98Trp and Gly99Trp U1A proteins, because we expected that comparison of the data from proteins labeled at two positions in helix C would contribute to a reliable assignment of the segmental dynamics of helix C. In addition, several anisotropy experiments were run on the Ala95Trp mutant and the results were similar to the Lys98Trp and Gly99Trp mutants, although not reported in detail since they were run at an earlier time using slightly different experimental methodologies.…”