Periodic Reporting for period 1 - MechHelicaseActiv8on (Unravelling the mechanism of eukaryotic helicase activation)
Reporting period: 2022-09-01 to 2024-08-31
The project aimed to unravel the mechanism of eukaryotic helicase activation in DNA replication initiation. During DNA replication, helicase is essential for unwinding the DNA double helix, a step critical for cellular division. However, the exact mechanisms by which the helicase is activated to start DNA unwinding, including the transition from double-stranded to single-stranded DNA, remain unclear.
Understanding DNA replication mechanisms is fundamental to advancing knowledge in genome maintenance. Errors in DNA replication can lead to genetic mutations, contributing to cancer and other genetic disorders. By shedding light on helicase activation, this research has the potential to aid in the development of targeted therapies for diseases that result from DNA replication errors.
The main objectives of the project were: (1) to uncover the fundamental mechanisms by which helicase is activated to initiate DNA unwinding, (2) to investigate the specific roles of various protein components within the helicase complex.
Understanding DNA replication mechanisms is fundamental to advancing knowledge in genome maintenance. Errors in DNA replication can lead to genetic mutations, contributing to cancer and other genetic disorders. By shedding light on helicase activation, this research has the potential to aid in the development of targeted therapies for diseases that result from DNA replication errors.
The main objectives of the project were: (1) to uncover the fundamental mechanisms by which helicase is activated to initiate DNA unwinding, (2) to investigate the specific roles of various protein components within the helicase complex.
For helicase to get activated during DNA replication initiation, the helicase has to transit from double stranded DNA to single stranded DNA. To find out the trajectory of lagging strand template during helicase activation, I purified all essential DNA replication proteins and confirmed their activity in reconstituted DNA replication assay. I prepared a set of helicase mutants required for testing the helicase activation mechanism.
Mcm10 assists helicase activation by supporting the transition from double stranded DNA to single stranded DNA and increasing replication speed. Together with our collaborators, we combined biochemistry with structural biology to further understand the mechanism of Mcm10 action. We identified distinct roles for the N-terminal (initiates replication) and C-terminal (increases speed) of Mcm10. Mutations in Mcm10 confirmed that its interaction with single stranded DNA is not essential for replication. We disseminated the findings by publishing in Nature Structural Biology with Open Acces.
Mcm10 assists helicase activation by supporting the transition from double stranded DNA to single stranded DNA and increasing replication speed. Together with our collaborators, we combined biochemistry with structural biology to further understand the mechanism of Mcm10 action. We identified distinct roles for the N-terminal (initiates replication) and C-terminal (increases speed) of Mcm10. Mutations in Mcm10 confirmed that its interaction with single stranded DNA is not essential for replication. We disseminated the findings by publishing in Nature Structural Biology with Open Acces.
The project contributed to our understanding of Mcm10 function as well as provided a separation-of-function mutant that might be useful for further research in the field. The helicase mutants might be further used in testing other processes, including how the helicase bypass DNA lesion. This could lead to further insight on not only the mechanism of DNA replication, but also other processes like DNA repair, which is important for genome stability.