Global conformational dynamics of a Y-family DNA polymerase during catalysis.
Replicative DNA polymerases are stalled by damaged DNA while the newly discovered Y-family DNA polymerases are recruited to rescue these stalled replication forks, thereby enhancing cell survival. The Y-family DNA polymerases, characterized by low fidelity and processivity, are able to bypass different classes of DNA lesions. A variety of kinetic and structural studies have established a minimal reaction pathway common to all DNA polymerases, although the conformational intermediates are not well defined. Furthermore, the identification of the rate-limiting step of nucleotide incorporation catalyzed by any DNA polymerase has been a matter of long debate. By monitoring time-dependent fluorescence resonance energy transfer (FRET) signal changes at multiple sites in each domain and DNA during catalysis, we present here a real-time picture of the global conformational transitions of a model Y-family enzyme: DNA polymerase IV (Dpo4) from Sulfolobus solfataricus. Our results provide evidence for a hypothetical DNA translocation event followed by a rapid protein conformational change prior to catalysis and a subsequent slow, post-chemistry protein conformational change. Surprisingly, the DNA translocation step was induced by the binding of a correct nucleotide. Moreover, we have determined the directions, rates, and activation energy barriers of the protein conformational transitions, which indicated that the four domains of Dpo4 moved in a synchronized manner. These results showed conclusively that a pre-chemistry conformational change associated with domain movements was too fast to be the rate-limiting step. Rather, the rearrangement of active site residues limited the rate of correct nucleotide incorporation. Collectively, the conformational dynamics of Dpo4 offer insights into how the inter-domain movements are related to enzymatic function and their concerted interactions with other proteins at the replication fork.
Department of Biochemistry, The Ohio State University, Columbus, Ohio, USA.
Cuiling
C
Xu
Brian A
BA
Maxwell
Jessica A
JA
Brown
Likui
L
Zhang
Zucai
Z
Suo
D002384:Catalysis; D020134:Catalytic Domain; D019951:DNA Polymerase beta / Q000737:chemistry*; D004260:DNA Repair; D004261:DNA Replication; D019641:DNA, Archaeal / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D011487:Protein Conformation; D013379:Substrate Specificity; D048229:Sulfolobus solfataricus / Q000737:chemistry
D019641:DNA, Archaeal; D019951:DNA Polymerase beta