Chromatin remodeler ALC1 prevents replication-fork collapse by slowing fork progression

Area of Science:

• Molecular Biology
• DNA Repair
• Chromatin Remodeling

Background:

• Amplified in liver cancer 1 (ALC1), also known as chromodomain helicase/ATPase DNA binding protein 1-like (CHD1L), is an SNF2 superfamily chromatin remodeler.
• ALC1 is involved in base-excision repair, with Poly(ADP-ribose) polymerase (PARP) signaling facilitating its recruitment to DNA damage sites.
• The precise role of ALC1 in DNA replication, particularly in response to replication-fork stress, remains to be fully elucidated.

Purpose of the Study:

• To investigate the critical role of ALC1 in regulating replication-fork progression in cleaved template strands.
• To analyze the functional relationship between ALC1 and Poly(ADP-ribose) polymerase 1 (PARP1) in response to DNA damage.
• To determine if ALC1's ATPase activity is essential for its function in camptothecin (CPT) tolerance.

Main Methods:

• Generation of ALC1-/-, PARP1-/-, and ALC1-/-/PARP1-/- chicken DT40 cell lines.
• Treatment of cell lines with camptothecin (CPT), a topoisomerase I poison, to induce replication-fork collapse.
• Assessment of CPT sensitivity, chromosome aberrations, replication-fork progression, and chromatin relaxation in wild-type and mutant cells.
• Introduction of an E165Q mutation in ALC1 to disrupt its ATPase activity.

Main Results:

• ALC1-/- and PARP1-/- cells showed increased sensitivity to CPT and a higher number of chromosome aberrations compared to wild-type cells.
• The phenotypes of ALC1-/- and PARP1-/- cells were similar, indicating ALC1's role in CPT tolerance is dependent on the PARP pathway.
• Inactivation of ALC1 led to a failure in slowing replication-fork progression after CPT exposure, highlighting ALC1's role at DNA-damage sites.
• ALC1-/E165Q cells exhibited CPT sensitivity comparable to null-mutant cells, suggesting ALC1's ATPase activity is critical for its function.
• CPT exposure induced chromatin relaxation in wild-type cells but not in ALC1-/- cells, supporting ALC1's role as a chromatin remodeler during replication.

Conclusions:

• ALC1 plays a critical, previously unappreciated role in regulating replication-fork progression at DNA-damage sites, particularly in response to CPT.
• ALC1 functions dependently on the PARP pathway for cellular tolerance to CPT-induced DNA damage.
• ALC1 likely acts as a chromatin remodeler, facilitating replication-fork slowing and maintaining genome stability during DNA replication stress.