複製蛋白DNA解旋酶的結構與功能探討揭示其易位模式及DNA解旋機制

Structural and functional studies of a replicative helicase GkDnaC reveal an insight into translocation and unwinding mechanism

羅羽華(Yu-Hua Lo)

指導教授 ： 孫玉珠 ； 蕭傳鐙

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2011年)

摘要

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.

關鍵字

DnaC 解旋酶 ； 複製叉 ； 解旋機製

並列摘要

無資料

並列關鍵字

DnaC helicase ； replication fork ； unwinding mechanism

參考文獻

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