Acute hydroxyurea-induced replication blockade results in replisome components disengagement from nascent DNA without...

Area of Science:

• Molecular Biology
• Cell Biology
• Genetics

Background:

• DNA replication forks face obstacles during S phase, leading to stalling and potential collapse.
• Cells possess mechanisms to maintain replication fork stability under replication stress.
• Understanding these mechanisms is crucial for cell survival and genome integrity.

Purpose of the Study:

• To investigate the mechanisms of replication fork stability maintenance in non-transformed human cells.
• To identify proteins associated with nascent DNA during replication stress.
• To elucidate the role of replisome components in fork stability and restart.

Main Methods:

• Isolation of proteins on nascent DNA (IPoND) coupled with mass spectrometry.
• Analysis of hTERT-RPE cells under various replication stress conditions (e.g., hydroxyurea).
• Assessment of replisome component association with chromatin and nascent DNA.

Main Results:

• Acute hydroxyurea treatment leads to single-stranded DNA accumulation and replisome disengagement from nascent DNA.
• The Cdc45-MCM-GINS helicase remains intact and associated with chromatin during acute stress.
• Sustained replication stress results in loss of replisome stability and impaired fork restart.
• Acute stress allows for fork restart despite replisome disengagement, preserving cellular competence.

Conclusions:

• Replication fork stability mechanisms differ between acute and sustained replication stress.
• The integrity of the Cdc45-MCM-GINS helicase is critical for maintaining fork stability under acute stress.
• Cells can disengage replisomes during acute stress while retaining the ability to restart replication, a process compromised by sustained stress.