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Researchers Use Mass Spec Method to ID Proteins Involved in DNA Repair


NEW YORK (GenomeWeb) – Using an unbiased proteomics analysis method, an international team of researchers has found that more than 90 proteins are involved in helping to repair damaged DNA before it is replicated.

"We believe this is the first global view of the protein landscape of a repair pathway," Markus Räschle, first author of the team's study, which was published last week in Science, told GenomeWeb. "We were not expecting it to be so complex, involving so many proteins."

In collaboration with teams from Harvard University and Niels Mailand's lab at the Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Räschle and other researchers from Matthias Mann's lab at the Max Planck Institute of Biochemistry in Germany used the Xenopus egg extract system in combination with mass spectrometry to develop CHROMASS — chromatin mass spectrometry — to find out what happens when replicative polymerases encounter DNA lesions during replication.

As they illustrated in their paper, they monitored the action of protein complexes on replicating chromatin containing DNA interstrand crosslinks (ICLs), which are particularly detrimental to DNA replication and can't be bypassed by the cell's replication machinery. As Xenopus egg extract is a model system for DNA replication, the team chose to use it by inserting small plasmids containing ICLs. They then used mass spectrometry to study the reaction.

"At the outset, we were not at all sure that we could monitor this repair pathway in a system-wide fashion, so initially we were very happy to see just a few factors," Räschle said. "But then after some improvements to the method, we quickly realized that it might be possible to comprehensively monitor the assembly and disassembly of repair complexes on the damaged DNA."

Not only did the researchers discover that ICLs trigger more than 90 DNA repair and genome maintenance proteins, they also found that the factors are specific to the type of damage present. "We were surprised by the selectivity of the method," Räschle said. "We predominantly saw enrichment of ICL repair factors on this particular DNA template, but we didn't find enrichment of factors involved in unrelated DNA repair pathways, such as non-homologous end joining or base excision repair."

Among the novel hits produced by their method, the team focused on two — SLF1 and SLF2 — which they were able to use to delineate a new pathway for the recruitment of SMC5/6, a protein important to genome maintenance. "Consistent with pivotal functions of SMC5/6 in the suppression of replication stress-induced, illegitimate recombination intermediates, depletion of SLF1 or SLF2 led to mitotic errors and compromised cell survival in response to genotoxic agents," they wrote in their paper.

Importantly, Räschle said, the CHROMASS approach the team developed can be used to study other chromatin-associated processes, such as the repair of other DNA lesions like double-strand breaks or protein-DNA crosslinks.

"One important goal is to use this unbiased approach to identify new DNA repair factors, in order to better understand the repair mechanisms and to perhaps explain the etiology of uncharacterized genetic disorders," he added. Ultimately, "our goal is to generate a comprehensive atlas of DNA repair factors and identify the pathways they are involved in."

Indeed, the team wrote in the paper's abstract, "The temporal profiles of hundreds of proteins across an extensive time course and a variety of perturbations provided a data-rich resource that could be mined to identify previously unknown genome maintenance factors."

Proteomics analysis could be used to study tumor samples from patients and identify the specific DNA repair pathway defects present, the researchers suggested.

"An atlas of the repair components would help us to do targeted experimentation in order to classify patients according to repair defects in their tumors," Räschle said. "For example, tumors with defects in interstrand crosslink repair could then be treated with drugs that are repaired by this pathway, whereas tumors with defects in end joining could be treated with a different class of chemotherapeutics."

The CHROMASS technique, and these types of findings, could also go a long way to bringing proteomics to the clinic, Räschle said. In the future, proteomic analysis could be used to study patient samples in real time, and to produce similar protein profiles, rather than identifying single proteins and attempting to use them as diagnostic markers. Such profiles of hundreds of thousands of proteins could help clinicians choose specific therapies for their patients.

"The work that we've been doing here lays the basis a little bit," he added, "but this is a further step down the road."