The SIOD disorder protein SMARCAL1 is an RPA-interacting protein involved in replication fork restart

Howard Hughes Medical Institute and Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Genes & development (Impact Factor: 10.8). 09/2009; 23(20):2415-25. DOI: 10.1101/gad.1832309
Source: PubMed


The integrity of genomic DNA is continuously challenged by the presence of DNA base lesions or DNA strand breaks. Here we report the identification of a new DNA damage response protein, SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), which is a member of the SNF2 family and is mutated in Schimke immunoosseous dysplasia (SIOD). We demonstrate that SMARCAL1 directly interacts with Replication protein A (RPA) and is recruited to sites of DNA damage in an RPA-dependent manner. SMARCAL1-depleted cells display sensitivity to DNA-damaging agents that induce replication fork collapse, and exhibit slower fork recovery and delayed entry into mitosis following S-phase arrest. Furthermore, SIOD patient fibroblasts reconstituted with SMARCAL1 exhibit faster cell cycle progression after S-phase arrest. Thus, the symptoms of SIOD may be caused, at least in part, by defects in the cellular response to DNA replication stress.

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Available from: Marie-Emilie Terret, Jan 30, 2015
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    • "HARP is a DNA replication stress response protein that is recruited to sites of DNA damage or stalled/arrested replication forks through its interactions with RPA which accumulates at the resultant ssDNA gaps present at these sites [5], [7], [8], [9]. HARP also appears to be associated with unperturbed replication forks [4]. "
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    ABSTRACT: HARP (SMARCAL1, MARCAL1) is an annealing helicase that functions in the repair and restart of damaged DNA replication forks through its DNA branch migration and replication fork regression activities. HARP is conserved among metazoans. HARP from invertebrates differs by the absence of one of the two HARP-specific domain repeats found in vertebrates. The annealing helicase and branch migration activity of invertebrate HARP has not been documented. We found that HARP from Drosophila melanogaster retains the annealing helicase activity of human HARP, the ability to disrupt D-loops and to branch migrate Holliday junctions, but fails to regress model DNA replication fork structures. A comparison of human and Drosophila HARP on additional substrates revealed that both HARPs are competent in branch migrating a bidirectional replication bubble composed of either DNA:DNA or RNA:DNA hybrid. Human, but not Drosophila, HARP is also capable of regressing a replication fork structure containing a highly stable poly rG:dC hybrid. Persistent RNA:DNA hybrids in vivo can lead to replication fork arrest and genome instability. The ability of HARP to strand transfer hybrids may signify a hybrid removal function for this enzyme, in vivo.
    Full-text · Article · May 2014 · PLoS ONE
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    • "The ATR-ATRIP complex, which interacts with the N-terminal OB fold of RPA70, was only able to bind RPA WT -ssDNA and not RPA t-11 -ssDNA (Figure 1B). In contrast, SMARCAL1, a protein that interacts with the RPA32 subunit of the RPA complex, was efficiently pulled down by both RPA WT -ssDNA and RPA t-11 -ssDNA (Figure 1B) (Bansbach et al., 2009; Ciccia et al., 2009; Yuan et al., 2009; Yusufzai et al., 2009). These results validate the capability of our approach to capture RPA-ssDNA binding proteins and identify the proteins that specifically interact with the N-terminal OB fold of RPA70, a key regulatory module for the DDR. "
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    ABSTRACT: PRP19 is a ubiquitin ligase involved in pre-mRNA splicing and the DNA damage response (DDR). Although the role for PRP19 in splicing is well characterized, its role in the DDR remains elusive. Through a proteomic screen for proteins that interact with RPA-coated single-stranded DNA (RPA-ssDNA), we identified PRP19 as a sensor of DNA damage. PRP19 directly binds RPA and localizes to DNA damage sites via RPA, promoting RPA ubiquitylation in a DNA-damage-induced manner. PRP19 facilitates the accumulation of ATRIP, the regulatory partner of the ataxia telangiectasia mutated and Rad3-related (ATR) kinase, at DNA damage sites. Depletion of PRP19 compromised the phosphorylation of ATR substrates, recovery of stalled replication forks, and progression of replication forks on damaged DNA. Importantly, PRP19 mutants that cannot bind RPA or function as an E3 ligase failed to support the ATR response, revealing that PRP19 drives ATR activation by acting as an RPA-ssDNA-sensing ubiquitin ligase during the DDR.
    Preview · Article · Dec 2013 · Molecular cell
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    • "Loss of SMARCAL1 function in cells causes the accumulation of collapsed replication forks and hyper-activation of the DNA damage response due to enzymatic action of the MUS81 structure-specific endonuclease (3,8). SMARCAL1 deficiency also causes increased sensitivity to replication stress agents like hydroxyurea (HU) and camptothecin, as well as an inability of stalled replication forks to recover DNA synthesis (3,4,6). Overexpression of SMARCAL1 causes replication-associated DNA damage, and lack of proper regulation causes fork collapse (3,15). "
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    ABSTRACT: SMARCAL1 promotes the repair and restart of damaged replication forks. Either overexpression or silencing SMARCAL1 causes the accumulation of replication-associated DNA damage. SMARCAL1 is heavily phosphorylated. Here we identify multiple phosphorylation sites, including S889, which is phosphorylated even in undamaged cells. S889 is highly conserved through evolution and it regulates SMARCAL1 activity. Specifically, S889 phosphorylation increases the DNA-stimulated ATPase activity of SMARCAL1 and increases its ability to catalyze replication fork regression. A phosphomimetic S889 mutant is also hyperactive when expressed in cells, while a non-phosphorylatable mutant is less active. S889 lies within a C-terminal region of the SMARCAL1 protein. Deletion of the C-terminal region also creates a hyperactive SMARCAL1 protein suggesting that S889 phosphorylation relieves an auto-inhibitory function of this SMARCAL1 domain. Thus, S889 phosphorylation is one mechanism by which SMARCAL1 activity is regulated to ensure the proper level of fork remodeling needed to maintain genome integrity during DNA synthesis.
    Full-text · Article · Oct 2013 · Nucleic Acids Research
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