Crystal structure of the Hda-sliding clamp complex provides insights into regulatory mechanism of prokaryotic replication initiation

Abstract

The topic of my thesis is about the bacterial DNA replication initiation regulatory mechanism. Bacterial DNA replication regulation is mainly focused on the initiator protein DnaA. ATP-bound DnaA is an active initiator of DNA replication in Escherichia coli. To inhibit the re-initiation during the progress of DNA replication, DnaA activity must be tightly controlled. Regulatory inactivation of DnaA (RIDA) is one of the major inhibitory events of the replication re-initiation, in which the Hda?sliding clamp complex loaded onto DNA directly interacts with the ATP-bound DnaA and stimulates the hydrolysis of ATP. To understand how the Hda associated with sliding clamp and how the complex promotes the ATP hydrolysis of DnaA, I determined the crystal structure of the E. coli Hda and sliding clamp complex at 3.23 A resolution. Hda and sliding clamp form an octamer, in which two pairs of Hda dimers are sandwiched by two clamps. Hda forms a tail-to-tail dimer, where the arginine finger of the Hda active site is blocked to restrict the access of DnaA. The structure provides insight into how the Hda?sliding clamp complex negatively controls its DnaA inhibitory activity during the replication, which is important for proper initiation by DnaA. Additionally, I worked on the Ssl1 protein that is a core component of general transcription factor TFIIH, which is involved in transcription and DNA repair. Through the mutational work on the several loops of Ssl1/p44, I could point out the Rad3/XPD interacting loop (β4-α5 loop) of Ssl1, and I modeled the active Rad3/XPD ? Ssl1/p44 complex. Also, I solved the crystal structure on the DNA double strand break repair protein complex Mre11 and Rad50 of Methanocaldococcus janaschii.