ScienceDaily (Jan. 1, 2010) — In the harsh judgment of natural selection, the ultimate measure of success is reproduction. So it's no surprise that life spends lavish resources on this feat, whether in the courtship behavior of birds and bees or replicating the cells that keep them alive. Now research has identified a new piece in an elaborate system to help guarantee fidelity in the reproduction of cells, preventing potentially lethal mutations in the process.
Stabilizing DNA. Researchers have identified the molecule SMARCAL1 as involved in cells' elaborate system for recognizing and repairing DNA damage during cell division. The protein is pictured above (green) in the presence of DNA (blue) as the chromosomes align along the mitotic spindle (red). (Credit: Image courtesy of Rockefeller University)
In experiments to be published in the December 18 issue of the Journal of Biological Chemistry, researchers at The Rockefeller University identified the molecule SMARCAL1 as part of cells' damage control response to malfunctioning DNA replication. In typical cell division, many different molecules have roles in guaranteeing the daughter strands of DNA are as identical as possible to their parent. Some molecules check for errors or 'proofread' the offspring for typos, for instance; others, when alerted to a problem, arrest the replication process and conduct repairs.
Lisa Postow, a postdoctoral fellow in Hironori Funabiki's Laboratory of Chromosome and Cell Biology, used mass spectroscopy to identify SMARCAL1 as involved in this intricate quality control process. Working with Brian T. Chait's Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, Postow found the protein in a proteomics screen for molecules that were drawn to a dangerous DNA repair problem called a double-strand break.
In both human cells and in cells from African clawed frog egg extract, Postow found that at double-strand breaks, SMARCAL1 gathered with another molecule called RPA, which is known to coat broken strands of DNA and protect them while damage is repaired. SMARCAL1 had an added interest, too: A mutation in the gene that produces it is involved in a rare but lethal disease called Schimke immuno-osseous dysplasia, a disorder that causes wide-ranging problems including kidney malfunction, immunodeficiency and growth inhibition.
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Journal Reference:
Postow et al. Identification of SMARCAL1 as a Component of the DNA Damage Response. Journal of Biological Chemistry, 2009; 284 (51): 35951 DOI: 10.1074/jbc.M109.048330
Identification of SMARCAL1 as a Component of the DNA Damage Response*
Lisa Postow‡,1, Eileen M. Woo‡§,2, Brian T. Chait§ and Hironori Funabiki‡,3
- Author Affiliations
From the ‡Laboratory of Chromosome and Cell Biology and
the §Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065
1 Supported by a fellowship from the Leukemia and Lymphoma Society of America. To whom correspondence should be addressed: 1230 York Ave., New York, NY 10065. Fax: 212-327-7292; E-mail: PostowL@rockefeller.edu.
Abstract
SMARCAL1 (also known as HARP) is a SWI/SNF family protein with an ATPase activity stimulated by DNA containing both single-stranded and double-stranded regions. Mutations in SMARCAL1 are associated with the disease Schimke immuno-osseous dysplasia, a multisystem autosomal recessive disorder characterized by T cell immunodeficiency, growth inhibition, and renal dysfunction. The cellular function of SMARCAL1, however, is unknown. Here, using Xenopus egg extracts and mass spectrometry, we identify SMARCAL1 as a protein recruited to double-stranded DNA breaks. SMARCAL1 binds to double-stranded breaks and stalled replication forks in both egg extract and human cells, specifically colocalizing with the single-stranded DNA binding factor RPA. In addition, SMARCAL1 interacts physically with RPA independently of DNA. SMARCAL1 is phosphorylated in a caffeine-sensitive manner in response to double-stranded breaks and stalled replication forks. It has been suggested that stalled forks can be stabilized by a mechanism involving caffeine-sensitive kinases, or they collapse and subsequently recruit Rad51 to promote homologous recombination repair. We show that depletion of SMARCAL1 from U2OS cells leads to increased frequency of RAD51 foci upon generation of stalled replication forks, indicating that fork breakdown is more prevalent in the absence of SMARCAL1. We propose that SMARCAL1 is a novel DNA damage-binding protein involved in replication fork stabilization.
Footnotes
↵2 Supported by a Howard Hughes Medical Institute predoctoral award.
↵3 Supported by the Rockefeller University and the Irma T. Hirschl/Monique Weill-Caulier Trust.
↵* This work was supported, in whole or in part, by National Institutes of Health Grants T32 CA09673 (to L. P.) and RR00862 (to B. T. C.).
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1 and S2.
↵5 L. Postow and H. Funabiki, unpublished data.
↵4 The abbreviations used are:
DSB double-stranded break
SIOD Schimke immuno-osseous dysplasia
ssDNA single-stranded DNA
RPA replication protein A
hRPA human RPA
MS mass spectrometry
dsDNA double-stranded DNA
MS/MS tandem MS
SB-DNA single biotin DNA
DB-DNA double biotin DNA
SH-DNA single hairpin DNA
HU hydroxyurea
PCNA proliferating cell nuclear antigen
Gy grays
ATR ATM and Rad3-related kinase
siRNA small interfering RNA
GFP green fluorescent protein.
Received July 23, 2009.
Revision received September 21, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.
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