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Detection of Post-Replicative Gaps Accumulation and Repair in Human Cells Using the DNA Fiber Assay

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Published on: February 3, 2022

The TIMELESS and PARP1 interaction suppresses replication-associated DNA gap accumulation.

Joanne Saldanha^1,2, Julie Rageul¹, Jinal A Patel¹

¹Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

Nucleic Acids Research

|May 27, 2024

Summary

TIMELESS (TIM) protein is crucial for coordinating DNA replication by ensuring proper Okazaki fragment processing and limiting DNA gaps. Its interaction with PARP1 is vital for DNA repair, presenting a potential cancer therapy target.

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Area of Science:

Molecular Biology
DNA Replication
Cancer Therapeutics

Background:

TIMELESS (TIM) protein stabilizes the replisome and ensures DNA replication fork progression.
The precise role of TIM in coordinating leading and lagging strand synthesis and limiting single-stranded DNA (ssDNA) exposure is not fully understood.

Purpose of the Study:

To elucidate the mechanism by which TIMELESS (TIM) coordinates DNA replication and limits ssDNA exposure.
To investigate the role of the TIM-PARP1 interaction in DNA repair pathways.
To explore the therapeutic potential of targeting TIM in cancer.

Main Methods:

Investigated the effects of TIM degradation on DNA replication and ssDNA gap formation.
Assessed the requirement of TIM for poly(ADP-ribosyl)ation in Okazaki fragment (OF) processing.
Examined the recruitment of XRCC1 to ssDNA gaps.
Studied the functional significance of the TIM-PARP1 complex disruption.

Main Results:

Acute TIM degradation at replication forks leads to ssDNA gaps due to defective Okazaki fragment (OF) processing.
TIM-deficient cells cannot support poly(ADP-ribosyl)ation, impairing OF processing by LIG1 and FEN1.
TIM-PARP1 complex disruption mimics TIM loss, highlighting its critical role in a compensatory DNA repair pathway.
Combined deficiency in FEN1 and TIM-PARP1 interaction causes synergistic DNA damage and cytotoxicity.

Conclusions:

TIM is essential for engaging PARP1 to the replisome, coordinating lagging strand synthesis with replication fork progression.
TIM acts as a synthetic lethal target in conjunction with OF processing enzymes, offering a potential strategy for cancer therapy.