Gene cloning and function analysis of replication factor C from Thermococcus kodakaraensis KOD1

Area of Science:

• Biochemistry
• Molecular Biology
• Genomics

Background:

• Replication factor C (RFC) is essential for DNA replication, facilitating the loading of proliferating cell nuclear antigen (PCNA) clamps onto DNA.
• PCNA clamps enable processive DNA synthesis by DNA polymerases.
• Understanding RFC function in archaea can provide insights into DNA replication mechanisms.

Purpose of the Study:

• To characterize the RFC homologue from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-RFC).
• To investigate the interaction and functional synergy between Tk-RFC, Tk-PCNA, and KOD DNA polymerase.

Main Methods:

• RFC-like genes from T. kodakaraensis KOD1 were cloned and co-expressed in E. coli.
• Purification of Tk-RFC subunits (Tk-RFCS and Tk-RFCL).
• Assays to measure the effect of Tk-RFC and Tk-PCNA on the DNA elongation rate of KOD DNA polymerase.

Main Results:

• Tk-RFC was successfully expressed and purified, consisting of small (Tk-RFCS) and large (Tk-RFCL) subunits.
• The presence of Tk-PCNA and Tk-RFC significantly increased the DNA elongation rate of KOD DNA polymerase.
• The DNA elongation rate was enhanced approximately 1.7-fold when Tk-RFC and Tk-PCNA were added at an equal molar ratio to KOD DNA polymerase.

Conclusions:

• Tk-RFC is a functional homologue of RFC involved in DNA replication in T. kodakaraensis.
• Tk-RFC, in conjunction with Tk-PCNA, enhances the processivity and elongation rate of the highly thermostable KOD DNA polymerase.
• This study contributes to understanding the molecular mechanisms of DNA replication in archaea.