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Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

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ATP bound to the origin recognition complex is important for preRC formation.

R D Klemm¹, S P Bell

¹Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

Proceedings of the National Academy of Sciences of the United States of America

|July 19, 2001

Summary

This summary is machine-generated.

The origin recognition complex (ORC) requires ATP hydrolysis for proper function. Hydrolysis-defective ORC mutants inhibit DNA replication by sequestering Cdc6p, suggesting ATP hydrolysis is crucial for pre-replicative complex disassembly.

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Area of Science:

Molecular Biology
Cell Cycle Regulation
DNA Replication

Background:

The origin recognition complex (ORC) binds replication origins and initiates DNA replication.
ORC binds and hydrolyzes ATP, but the role of hydrolysis remains unclear.
ATP binding to Orc1p enhances DNA binding, yet hydrolysis function is unknown.

Purpose of the Study:

To investigate the function of ATP hydrolysis by the origin recognition complex (ORC).
To elucidate the role of ATP hydrolysis in the assembly and disassembly of the prereplicative complex (preRC).

Main Methods:

Generation of dominant lethal Orc1p mutants defective in ATP hydrolysis but not binding.
Overexpression of mutant ORC and wild-type/mutant Cdc6p to assess phenotypic suppression.
Analysis of ORC's role in prereplicative complex formation and cell growth inhibition.

Main Results:

Hydrolysis-defective ORC mutants were generated and exhibited dominant lethality.
These mutants inhibited prereplicative complex formation upon overexpression.
Overexpression of wild-type Cdc6p suppressed the dominant lethal phenotype, unlike mutant Cdc6p.

Conclusions:

ATP hydrolysis by ORC is essential for cell viability and proper DNA replication.
Hydrolysis-defective ORC mutants likely inhibit growth by sequestering Cdc6p.
Cdc6p recognition of ATP-bound Orc1p and coupled ATP hydrolysis to preRC disassembly are proposed mechanisms.