Response of the bacteriophage T4 replisome to noncoding lesions and regression of a stalled replication fork

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA damage is a constant threat to genome integrity.
• Replication fork progression can be halted by DNA lesions, potentially leading to fork collapse.
• Understanding how replication machinery handles DNA lesions is crucial for DNA repair mechanisms.

Purpose of the Study:

• To investigate the impact of noncoding DNA lesions on the bacteriophage T4 replisome.
• To differentiate the effects of lesions on the leading versus lagging strand templates.
• To elucidate the molecular mechanisms underlying replication fork stability and collapse.

Main Methods:

• Utilized bacteriophage T4 replisome system.
• Introduced noncoding DNA lesions into leading and lagging strand templates.
• Observed replication fork progression and polymerase activity in response to lesions.

Main Results:

• Lesions in the lagging strand template did not impede replication fork progression.
• Stalled lagging strand polymerase recycled and initiated new Okazaki fragment synthesis.
• Leading strand template lesions halted replication fork progression approximately 1 kb beyond the lesion.
• Replication fork collapse occurred with leading strand lesions, despite continued primosome and lagging strand polymerase activity.
• A DNA repair helicase, UvsW, processed the stalled fork into a 'chicken-foot' structure.

Conclusions:

• The bacteriophage T4 replisome exhibits distinct responses to DNA lesions on leading versus lagging strands.
• Lagging strand lesions are managed through polymerase recycling and Okazaki fragment restart.
• Leading strand lesions trigger replication fork collapse and subsequent processing by DNA repair helicases like UvsW.
• The 'chicken-foot' structure may represent an intermediate in an error-free lesion bypass pathway.