Sci. Aging Knowl. Environ., 19 March 2003
DNA repair might depend on patterns of histone modifications
R. John Davenporthttp://sageke.sciencemag.org/cgi/content/full/sageke;2003/11/nw45
Key Words: H2AX phosphorylation acetylation
SANTA FE, NEW MEXICO--Like "HELP" scrawled in the snow by stranded skiers, marks on a protein near chromosome breaks draw molecular rescue workers to the trouble. This conclusion comes from research presented here at the Enzymology of Chromatin and Transcription Keystone Symposium on 11 March 2003.
Severing DNA with radiation or chemicals can cause trouble. Left unmended, the breaks can spur cancer-causing mutations, and inherited defects in the cellular repair machinery underlie diseases that resemble premature aging (see Fry Review, "Of Hyperaging and Methuselah Genes," "Laid to Waste," "Deadly Fate," and "Deadly Network"). In cells, DNA compacts into so-called chromatin by winding tightly around protein spools called histones, and chromatin must loosen for repair machinery to access damage. According to previous work, restoration proceeds only if the protein histone H2A carries a phosphate group on one particular amino acid. However, restructuring chromatin for other processes--such as turning on genes--depends on chemical changes to numerous amino acids in histones, so molecular biologists John Moore and Jocelyn Krebs of the University of Alaska, Anchorage, investigated whether DNA repair also requires chemical enhancements at additional sites.
The researchers altered the yeast gene that encodes H2A to create proteins containing the unmodifiable amino acid alanine in place of one of several amino acids that often receive phosphate or acetyl groups. They inserted each new gene into yeast cells, exposed the cells to the DNA-damaging chemical methyl methanesulfonate (MMS), and measured survival. Tweaking any one of three particular amino acids in H2A rendered cells unusually sensitive to MMS treatment. The results indicate that those amino acids are crucial for repair, perhaps because the cell modifies them.
Cells use two mechanisms to fix breaks. One, homologous repair, employs duplicate sequences from another chromosome to guide the stitching; the other, nonhomologous end joining (NHEJ), glues broken strands together without a matching sequence. The MMS survival test doesn't distinguish between the two mechanisms, so the researchers also performed a standard NHEJ test. They determined how efficiently yeast cells joined the ends of a test piece of linear DNA to create a circular molecule. Changes to two of the amino acids that sensitized cells to MMS diminished the cells' ability to seal the DNA. Swapping another did not impair end joining, however, suggesting that it is crucial only for homologous repair. Altering any one of three lysines--an amino acid that often accepts acetyl groups--also diminished NHEJ. Additional experiments suggest that repair depends on modification of the lysines: Changing one lysine to an amino acid that mimics the shape of a lysine bearing an acetyl group maintained a cell's NHEJ capacity. The results suggest that a unique set of chemical changes to histones, including both phosphate and acetyl additions, attracts the NHEJ machinery. The team plans to investigate whether the pinpointed amino acids are modified after DNA damage.
A second study presented here identifies an enzyme that might make such chemical changes. To explore how the phosphate on H2A enables repair, molecular biologist Jacques C�t� of Laval University in Quebec, Canada, and colleagues sought proteins that bind to it. A gang of proteins known to tack acetyl groups onto histones stuck to the modification on a fragment of H2A. Furthermore, the proteins flocked to sites of DNA breaks, and cells that lack members of the conglomeration didn't survive MMS treatment. The researchers propose that the multiprotein enzyme, once bound, makes further modifications. Perhaps, they speculate, these activities open up chromatin structure and permit repair.
The studies suggest that fixing DNA breaks requires modifications to multiple amino acids in H2A, says molecular biologist Shelley Berger of the Wistar Institute in Philadelphia. Different patterns of modification might specify different DNA-mending processes, she adds. C�t� plans to address whether histone-modifying enzymes serve only to make DNA accessible or whether they participate directly in repair. Future studies will provide additional insight into how the histone SOS attracts the emergency crew.
--R. John Davenport
March 19, 2003
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