DNA helicases are motor proteins that play essential roles in DNA replication, repair and recombination. The fundamental reaction of replicative hexameric helicase is the unwinding of duplex DNA. However, our understanding of how helicase unwinds remains vague due to insufficient structural information. Here, we report two crystal structures of the DnaB-family replicative helicase from Geobacillus kaustophilus HTA426 (GkDnaC) in the apo-form and in a single-stranded DNA (ssDNA) bound form. The GkDnaC–ssDNA complex structure reveals that three symmetrical basic grooves on the interior surface of the hexamer individually encircle ssDNA. The ssDNA-binding pockets in this structure are directed toward the N-terminal domain collar of the hexameric ring, thus orienting the ssDNA toward the DnaG primase to facilitate the synthesis of short RNA primers. These findings provide insights into the mechanism of ssDNA binding and provide a working model to establish a novel mechanism for DNA translocation at the replication fork. In addition, several key residues responsible for DNA-binding may play a role in DNA translocation during the unwinding process. We also studied these GkDnaC mutants by helicase assays, ATPase assays and single-molecule tethered particle motion (smTPM) experiments. The results demonstrated that the helicase activities of these GkDnaC mutants are 2~4 fold higher that of the wide-type protein. However, the enhancement of helicase activity from mutant proteins is not contributed by the efficiency of ATP hydrolysis, but by the helicase/ssDNA interaction. It implies that losing partial interaction with ssDNA leads to faster DNA translocation. We also studied whether accessory proteins affect helicase behaves at the junction. Taken together, our results reveal an insight into DNA unwinding mechanism.

1 Abstract in Chinese………………………………….....………..…………………..1
2 Abstract……………………………......……………...……………………………....2
3 Introduction…………………………………………..……………..…………………3
4 Materials and methods
4.1 Cloning and mutagenesis ...................………..…………………...……………6
4.2 Gel-filtration chromatography…………………….………………………….…...7
4.3 Crystallization, data collection and structure determination……………......7
4.4 Surface Plasmon Resonance (SPR)…………………………………….................8
4.5 ATP hydrolysis assay…………………………………………………....................9
4.6 EMSA helicase assay………………………………………………...…................10
4.7 Single molecule Tethered Particle Motion (smTPM)
4.7.1 DNA substrates design……………………………………………..…………….10
4.7.2 Helicase unwinding experiments……………………………………………….11 4.7.3 Imaging acquisition and analysis…………………............…….……………12
4.7.4 Bootstrap estimation for Unwinding rates…………………………………...12
5 Results
5.1 Crystal structure of GkDnaC and its complex with ssDNA…………....……13
5.2 Characteristics of GkDnaC bound to ssDNA……….....…..…………….…….14
5.3 Mutagenesis studies of GkDnaC–ssDNA interaction sites by SPR.............15
5.4 The role of flanked DNA binding loop I.…....……..….…...........................16
5.5 GkDnaC form a stable complex with GkDnaI…………………...………....…16
5.6 GkDnaI facilitates ssDNA binding when in complex with GkDnaC………..17
5.7 Helicase activity alters as helicase loses partial DNA-binding interaction....................................................................................................18
5.8 Roles of primosomal proteins in helicase unwinding…………............…...20
5.9 Helicase unwinding process monitored by smTPM…………….......……….21
5.10 ATP dependence of GkDnaC unwinding rate.……...…………..................22
5.11 The strength of Helicase-DNA interaction is correlated with DNA unwinding rates……………………………….........……………………………..……..22
5.12 Primase accelerates the unwinding rate of GkDnaC helicase..…………...23
6 Discussion
6.1 Nucleotide-binding site neighboring with DNA-interaction site..…..……25
6.2 Structural comparison of GkDnaC with papillomavirus E1 helicase…..…..25
6.3 A model for ssDNA binding in the N-terminal region during DNA translocation................................................................................................26
6.4 Helicase translocation and unwinding mechanism……........……………....27
7 Figures………………………………………...……………………........................30
8 Tables………………………………….....................……...……………………….69
9 Reference………………...………………….......................…………................75

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