SMARCAL1 and replication stress: an explanation for SIOD?

Abstract

The SNF2 family of ATPases acts in the context of chromatin to regulate transcription, replication, repair and recombination. Defects in SNF2 genes cause many human diseases. For example, mutations in SMARCAL1 (also named HARP) cause Schimke immuno-osseous dysplasia (SIOD); a multi-system disorder characterized by growth defects, immune deficiencies, renal failure and other complex phenotypes. Several groups including ours recently identified SMARCAL1 as a replication stress response protein. Importantly, SMARCAL1 localizes to stalled replication forks and this localization of SMARCAL1 activity prevents DNA damage accumulation during DNA replication. We determined that SIOD-related SMARCAL1 mutants could not prevent replication-associated DNA damage in cells in which endogenous SMARCAL1 was silenced, establishing the first link between SIOD and a defect in a specific biological activity. Here, we also report that cells from patients with SIOD exhibit elevated levels of DNA damage that can be rescued by re-introduction of wild-type SMARCAL1. Our data suggest that loss of SMARCAL1 function in patients may cause DNA replication-associated genome instability that contributes to the pleiotropic phenotypes of SIOD.

Keywords: ATM; ATR; DNA damage response; DNA replication; DNA-PK; HARP; RPA; SMARCAL1; checkpoint; replication fork.

Figure 1

SIOD patient fibroblasts exhibit increased DNA damage signaling. (A) SIOD patient-derived human fibroblasts stably expressing vector only, WT, ΔN, or R764Q SMARCAL1 were cultured on coverslips and stained with antibodies to γH2AX and appropriate secondary antibodies. The percentage of cells with γH2AX staining was scored; error bars are standard deviation 300 cells were counted with each cell type analyzed in triplicate. (B) Cells with an integrated empty vector (Vector) or vector expressing wild-type SMARCAL1 (WT) were left untreated (NT) or treated with 2 mM hydroxyurea (HU) for 5 h then released into normal growth media for 0 or 8 h as indicated. Total cell lysates were prepared and immunoblotted with antibodies to SMARCAL1, phosphorylated RPA2, or total RPA2. (C) Model for SMARCAL1 regulation at stalled replication forks. DNA polymerase and helicase activities are uncoupled at replication forks in response to DNA lesions or agents like HU that interfere with DNA synthesis. Uncoupling causes the formation of ssDNA that is rapidly coated with RPA. An interaction between the N-terminus of SMARCAL1 and RPA2 localizes SMARCAL1 to the stalled fork where it is phosphorylated by DNA-damage activated kinases.